Benzotriazine Oxides as Drugs Targeting Mycobacterium Tuberculosis

ABSTRACT

Benzotriazine doxides are disclosed as drugs targeting  mycobacterium tuberculosis , including novel compounds of formula I:

This application is a continuation of Ser. No. 13/726,135, filed Dec.23, 2012; which claims priority to PCT/US12/68636, filed, Dec. 7, 2012,which claims priority to U.S. 61/567,829 filed Dec. 7, 2011.

This invention was made with government support under NIH Contract No.HHSN266200600011C and NIH Grant No. 1R56AI09081-017 awarded by NationalInstitutes of Health (NIH); the Government has certain rights in thisinvention.

INTRODUCTION

The current R&D pipeline for new tuberculosis (TB) drugs is inadequateto address emerging drug-resistant strains. Accordingly, filling theearly drug development pipeline with novel therapies likely to slowadditional drug resistance is urgent. Combination chemotherapy has beena standard of care for TB since the 1950s, when it was shown thatcombining drugs slowed the development of drug resistance, particularlyfor bactericidal compounds.1 The next major breakthrough in TB treatmentwas the inclusion of rifampin and pyrazinamide in the multidrug cocktailbecause those drugs significantly accelerated the clearance of theinfection, presumably through their bactericidal activity against thepopulations of bacterial persisters. The currently recommended DirectObserved Therapy Short-course (DOTS) regimen, has been shown to behighly effective in treating drug-sensitive TB, but requires at least 6months of treatment. The next major breakthrough in the treatment of TBis likely to be the development of an improved regimen that requiresless treatment time, thus decreasing cost, increasing patientcompliance, and slowing the emergence of multidrug-resistant TB (MDR-TB)strains.

Mycobacterium tuberculosis (Mtb) survival in stages of nonreplicatingpersistence (NRP) that are tolerant to many drugs provides a possibleexplanation for the long treatment regimens required to eliminate aninfection.2 The development of new combination therapy regimens,including drugs that are bactericidal against these NRP stages of Mtb,has the potential for shortening the length of treatment regimens.Metronidazole, a 5-nitroimidazole antibiotic primarily used for thetreatment of anaerobic bacterial infections, was one of the firstcompounds shown to be bactericidal against NRP Mtb.2 Two newer5-nitroimidazoles, PA-8243 and OPC-676834, have greater potenciesagainst Mtb and are being evaluated clinically in combinations withstandard TB drugs. So far, both of these 5-nitroimidazoles have beenshown to have early bactericidal activity in patients.5,6 Preclinicalstudies in murine models of TB indicate that including bactericidaldrugs with activity against NRP Mtb in a combination regimen shortensthe time required to cure mice of the infection,7 but it remains to bedetermined whether these findings will translate to the ability toshorten the length of clinical treatment regimens.

To develop new drugs that shorten TB treatment, we evaluated additionalclasses of bioreductively-activated compounds with previously reportedantimicrobial activities for their activities against Mtb H37Rv. Wedisclose BTOs as a new class of antitubercular compounds with manyunique properties that make them well-suited as anti-TB drugs,particularly with the potential to shorten the length of treatment.

Relevant literature includes: Chopra et al, J Med Chem. 2012 Jul. 12;55(13):6047-60; Hay, et al., J. Med. Chem., 2008, 51 (21), pp 6853-6865;Zeman, et al., Int. J. Radiat. Oncol., Biol., Phys. 1989, 16, 977-981;Minchinton, et al, Int. J. Radiat. Oncol., Biol., Phys 1992, 22,701-705; Kelson, et al., Anti-Cancer Drug Des. 1998, 13, 575-592; Jiang,et al., Bioorg. Med. Chem. Lett. 2006, 16, 4209-4213; and Jiang, et al,Arch. Pharm. (Weinheim, Ger.) 2007, 340, 258-263.

SUMMARY OF THE INVENTION

In one embodiment the invention provides compounds having the structureof formula I:

wherein: each X is independently H, halogen, alkyl, OR, SR, NR′R, BR′R,heterocycles, or another functional group, wherein each R isindependently H, halogen, alkyl, or another functional group; W is N, C,O, S, H or B or another linking atom; each A and B is H or optionallysubstituted alkyl, which may be joined in an optionally hetero-,cycloalkyl; and Z is an optionally present, optionally substituted 4-8membered ring, saturated or unsaturated, fused to the bezotriazine ringat either the 6,7-, 5,6- or 7,8-position, or a pharmaceuticallyacceptable salt or stereoisomer thereof.

In particular embodiments each X is independently H, halogen, alkyl, OR,SR, NR′R, BR′R, wherein each R is independently H, halogen or alkyl,more particularly H; W is N, C, O, S, or B, more particularly N; each Aand B is H or optionally substituted alkyl, which may be joined in anoptionally hetero-, cycloalkyl, more particularly an optionallysubstituted piperidinyl or pyrrolidinyl; and Z is an optionally present,optionally substituted 4-8 membered ring, saturated or unsaturated,fused to the bezotriazine ring at either the 6,7-, 5,6- or 7,8-position,particularly an optionally present, optionally substituted 5 or6-membered ring, saturated or unsaturated, fused to the bezotriazinering at the 6,7-position, more particularly a present, 5-membered ring,unsaturated, fused to the bezotriazine ring at the 6,7-position.

In particular embodiments:

each X is independently H, halogen, alkyl, OR, SR, NR′R, or BR′R,wherein each R is independently H, halogen, or alkyl; W is N, C, O, S orB; each A and B is H or optionally substituted alkyl, which may bejoined in an optionally hetero-, cycloalkyl; and Z is an optionallysubstituted 4-8 membered ring, saturated or unsaturated, fused to thebezotriazine ring at either the 6,7-, 5,6- or 7,8-position.

each X is independently H, halogen, alkyl, OR, SR, NR′R, or BR′R,wherein each R is independently H, halogen, or alkyl; W is N, C, O, S orB; each A and B is optionally substituted alkyl, which may be joined inan optionally hetero-, cycloalkyl; and Z is an optionally substituted4-8 membered ring, saturated or unsaturated, fused to the bezotriazinering at either the 6,7-, 5,6- or 7,8-position.

each X is independently H, halogen, alkyl, OR, SR, NR′R, or BR′R,wherein each R is independently H, halogen, or alkyl; W is N; each A andB is optionally substituted alkyl, which may be joined in aheterocycloalkyl; and Z is an optionally substituted 5 or 6-memberedring, saturated or unsaturated, fused to the bezotriazine ring at the6,7-position;

each X is H; W is N; A and B are joined in an optionally substitutedpiperidinyl or pyrrolidinyl; and Z is a 5-membered ring, fused to thebezotriazine ring at the 6,7-position.

In particular embodiments the compound is a BTO of a particularlyrecited structure disclosed herein, such as in the tables below.

In other embodiments the invention provides pharmaceutical compositionsand kits comprising a subject BTO compound and a second, differentanti-mycobacterium tuberculosis (Mtb) drug.

The invention also provides methods of making subject BTO compoundscomprising an oxidation reaction using HOF:ACN.

The invention also provides methods of treating a mycobacteriumtuberculosis (Mtb) infection, comprising: contacting a person in needthereof with an effective amount of a subject compound or composition.

In another aspect the invention provides methods method of treating amycobacterium tuberculosis (Mtb) infection, comprising: contacting aperson in need thereof with an effective amount of a 1,2,4-benzotriazinedi-N-oxide (BTO).

The subject methods of use or treatment may further comprise asubsequent step of detecting a resultant diminution in the infectionand/or a prior step of detecting the infection.

In particular embodiments, the infection comprises nonreplicatingpersistence (NRP) Mtb cells.

The invention encompasses all combinations of particular and preferredembodiments as though each had been laboriously, separately recited.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Plasma drug concentrations of 8a and 20q in female Balb/c mice(n=3 mice per time point) after po administration of 100 mg/kg of eachcompound. Blood was collected at time points through 24 hr post-dose,processed to plasma and analyzed for each of the test compounds. Theconcentrations at the 24 hr time point for both compounds was lower thanthe limit of quantitation.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments and examples areprovided by way of illustration and not by way of limitation. Thoseskilled in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results. Unless contraindicated or noted otherwise, in thesedescriptions and throughout this specification, the terms “a” and “an”mean one or more, the term “or” means and/or and polynucleotidesequences are understood to encompass opposite strands as well asalternative backbones described herein. Furthermore, genuses are recitedas shorthand for a recitation of all members of the genus; for example,the recitation of (C1-C3) alkyl is shorthand for a recitation of allC1-C3 alkyls: methyl, ethyl and propyl, including isomers thereof.

The invention provides compounds of formula I, and subgenera and speciesthereof, compositions, including formulations and kits, containing suchcompounds, and methods of making and using the disclosed compounds andcompositions.

The invention also provides novel methods for preparing these compoundsby the oxidation of the compounds using HOF.ACN:

Reagents and Conditions: a) HOF.ACN, DCM, −15° C., 30 min. b) HNR₂R₃,Et₃N, DCM.

The activity of these compounds against M. tuberculosis has beendetermined using a minimum inhibitory concentration (MIC) measurement.The toxicity and selectivity of the compounds have been quantified by a50% cytotoxicity concentration (CC₅₀) using a Vero cell viability assaywith the Promega Cell Titer Glo reagent. The activity againstnon-replicating M. tuberculosis has been measured using the publishedLow-Oxygen Recovery Assay (LORA). The Selectivity Index (SI) of eachcompound was measured as the ratio of the CC₅₀/TB-MIC values.

TABLE P1 Anti-TB activity of side chain substituted BenzotriazineOxides. VERO Tox TB MIC- LORA Compound TB MIC-SRI (CC₅₀) UIC MIC SISRI-003356 1.25 SRI-012255 2.5 20.7 1.82 1.76 8.3 SRI-012256 2.5 7.91.64 0.5 3.2 SRI-012257 0.625 2.3 0.22 <0.125 3.7 SRI-012258 5.0 26.31.95 0.99 5.3 SRI-012259 1.25 4.1 0.52 0.38 3.3 SRI-012260 1.25 8.9 0.940.84 7.1 SRI-012261 1.25 8.9 0.92 0.36 7.1 SRI-012262 2.5 21.6 2.0 1.288.6 SRI-012263 2.5 11.7 1.98 0.83 4.7 SRI-012264 0.625 3.4 0.6 0.42 5.5SRI-012265 2.5 10.4 3.58 0.68 4.2 SRI-012266 2.5 8.9 3.77 0.75 3.6SRI-012267 5.0 21.0 1.93 0.98 4.2 SRI-012268 5.0 20.2 2.43 1.0 4.0SRI-012269 2.5 7.3 1.91 2.33 2.9 SRI-012270 1.25 5.2 0.57 0.39 4.2SRI-012271 5.0 36.5 3.63 1.94 7.3 SRI-012272 10.0 50.0 6.57 4.87 5.0SRI-012273 1.25 4.6 0.48 0.48 3.7

TABLE P2 Anti-TB activity of ring substituted Benzotriazine Oxides. TBTB MIC- VERO MIC- LORA Compound SRI Tox-SRI UIC MIC SI SRI-012274 0.3123.5 0.93 2.80 11.4 SRI-012275 0.625 <2.5 0.68 0.47 <4 SRI-012276 0.3122.5 0.40 0.88 8.0 SRI-012277 0.312 2.3 0.33 0.34 7.3 SRI-012278 5 6.32.00 1.87 1.3 SRI-012279 1.25 4.4 0.49 0.71 3.5 SRI-012280 0.312 <2.5<0.125 0.21 <4 SRI-012281 1.25 <2.5 0.55 0.40 <4 SRI-012282 0.625 <2.50.39 0.21 <4 SRI-012283 1.25 <2.5 0.50 0.42 <4 SRI-012284 0.625 4.6 0.350.75 7.3 SRI-012285 2.5 15.8 15.14 16.16 6.3 SRI-012286 1.25 36.0 0.950.83 28.8 SRI-012301 0.625 1.36 0.21 0.22 2.2 SRI-012302 0.156 1.6<0.125 <0.125 10.3 SRI-012303 0.625 2.62 0.94 0.63 4.2 SRI-012304 1.252.6 1.46 0.78 2.1 SRI-012305 0.156 <0.125 0.20 SRI-012306 1.25 2.65<0.125 0.41 2.1 SRI-012396 1.25 SRI-012397 0.625 SRI-012398 2.5SRI-012399 2.5 SRI-012401 0.312 SRI-012402 0.312 SRI-012403 0.625 33.653.8 SRI-012404 1.25 50 40 SRI-012405 0.312 21.57 69.1 SRI-012405 0.62522.5 36.0 SRI-012459 0.625 21.8 34.9 SRI-012460 0.312 21.8 69.9SRI-012461 0.156 2.3 14.7 SRI-012462 0.625 SRI-012463 0.625 9.85 15.8SRI-012464 0.625 SRI-012465 0.156 SRI-012466 0.156 SRI-012467 0.312 0.812.6 SRI-012468 2.5 100 40 SRI-012469 2.5 50 20.0 SRI-012470 5 50 10.0SRI-012473 0.312 3.7 11.9 SRI-012496 1.25 21.78 17.4 SRI-012497 1.25 5040.0 SRI-012498 1.25 50 40.0

TABLE P3 Anti-TB activity of di-N-alkyl Benzotriazine Oxides. TB TB MIC-MIC- LORA VERO Compound SRI UIC MIC Tox SI SRI-003270 2.5 5.4 2.2SRI-009728 0.312 12.4 39.6 SRI-012495 0.312 17.42 55.8 SRI-012530 0.625SRI-012562 0.625 SRI-012563 0.625 SRI-012564 0.312 SRI-012565 0.312SRI-012566 5 SRI-012567 1.25 SRI-012568 5

TABLE P4 Activity of Compounds Against Drug-Resistant M. tuberculosisstrains. Mtb Strains Resistant to P-aminosalicyclic Streptomycin acidIsoniazid Kanamycin Ethionamide Ethambutol Compounds H37Rv ATCC 35820ATCC 35821 ATCC 35822 ATCC 35827 ATCC 35830 ATCC 35837 SRI-003270 1.250.31 1.25 1.25 1.25 0.31 0.63 SRI-003274 1.25 1.25 1.25 1.25 1.25 1.251.25 SRI-012495 0.31 0.02 0.62 0.31 0.31 0.31 0.31 SRI-012496 1.25 1.251.25 1.25 1.25 1.25 1.25 SRI-012497 1.25 1.25 1.25 2.5 2.5 1.25 2.5SRI-012498 1.25 1.25 1.25 2.5 1.25 1.25 1.25 SRI-009728 0.31 0.31 0.310.31 0.31 0.16 0.31 SRI-012530 0.62 0.16 0.31 0.31 0.63 0.62 0.62SRI-012403 0.62 0.31 0.62 1.25 1.25 0.62 0.62 SRI-012405 0.62 0.16 0.310.62 0.62 0.31 0.62 SRI-012286 1.25 0.63 1.25 2.5 1.25 1.25 1.25SRI-003356 2.5 1.25 1.25 2.5 2.5 1.25 2.5 SRI-012562 0.62 0.15 0.31 0.150.31 0.31 0.31 SRI-012563 0.62 <0.07 0.15 <0.07 0.31 0.15 0.31SRI-012564 0.31 <0.07 0.07 <0.07 0.15 0.07 0.15 SRI-012565 0.31 <0.070.15 0.07 0.15 0.15 0.31 SRI-012566 5 2.5 2.5 2.5 2.5 0.62 2.5SRI-012567 1.25 0.31 0.62 0.31 0.62 0.31 0.62 SRI-012568 5 2.5 2.5 2.52.5 0.62 2.5

TABLE P5 Representative Structures of Compounds. Compound SRI-003356

SRI-012255

SRI-012256

SRI-012257

SRI-012258

SRI-012259

SRI-012260

SRI-012261

SRI-012262

SRI-012263

SRI-012264

SRI-012265

SRI-012266

SRI-012267

SRI-012268

SRI-012269

SRI-012270

SRI-012271

SRI-012272

SRI-012273

SRI-012274

SRI-012275

SRI-012276

SRI-012277

SRI-012278

SRI-012279

SRI-012280

SRI-012281

SRI-012282

SRI-012283

SRI-012284

SRI-012285

SRI-012286

SRI-012301

SRI-012302

SRI-012303

SRI-012304

SRI-012305

SRI-012306

SRI-012396

SRI-012397

SRI-012398

SRI-012399

SRI-012401

SRI-012402

SRI-012403

SRI-012404

SRI-012405

SRI-012405

SRI-012459

SRI-012460

SRI-012461

SRI-012462

SRI-012463

SRI-012464

SRI-012465

SRI-012466

SRI-012467

SRI-012468

SRI-012469

SRI-012470

SRI-012473

SRI-012496

SRI-012497

SRI-012498

SRI-003270

SRI-009728

SRI-012495

SRI-012530

SRI-012562

SRI-012563

SRI-012564

SRI-012565

SRI-012566

SRI-012567

SRI-012568

TABLE E1 Additional BTO Activity Data Compound Cmpd # Reference WeightStructure  1 SRI-013330 368.4386

 2 SRI-013329 323.3543

 3 SRI-013328 284.3174

 4 SRI-013327 366.3941

 5 SRI-013326 354.3831

 6 SRI-013310

 7 SRI-013277 351.3792

 8 SRI-013253 436.5092

 9 SRI-013243 300.36 

10 SRI-013158 312.371 

11 SRI-012722 390.3633

12 SRI-012721 352.392 

13 SRI-012720 366.3941

14 SRI-012621 340.3563

15 SRI-012620 382.8491

16 SRI-012619 408.4556

17 SRI-012618 366.3941

18 SRI-012616 408.4556

MP MP MP MP MIC MIC MIC MIC MP MTT % MP MTT % CFU MTT % H37Rv MBC INH-rRMP-R OFX-R CFU red. viable CFU red. viable red. viable Cmpd (ug/ml)H37Rv (ug/ml) (ug/ml) (ug/ml) LORA 0.01 ug/ml 0.01 ug/ml 0.1 ug/ml 0.1ug/ml 1 ug/ml 1 ug/ml 1 4.6 2 1.0 3 0.4 4 4.6 5 4.4 6 0.4 7 17.6 8 0.7 90.5 10 0.391 1.56 0.391 0.098 0.195 bad curve 1.49 103 1.27 94 1.52 10711 0.098 0.781 0.195 0.024 0.024 3.125 (bad 1.5 113 1.66 108 3.79 104curve) 12 0.391 1.56 0.098 0.098 0.195 1.56 (bad 1.31 121 1.41 94 0 88curve) 13 0.098 0.781 0.098 0.049 0.024 bad curve 1.34 161 1.04 123 2.87102 14 0.195 0.781 0.098 0.049 0.098 0.391 0.8 112 0.79 82 3.08 1 150.098 1.56 0.098 0.098 0.024 bad curve 1.21 102 0.92 75 2.67 58 16 0.1953.125 0.098 0.195 0.098 bad curve 1.72 122 1.01 97 0.35 108 17 0.0980.781 0.049 0.027 0.049 0.391 1.16 104 0.89 82 2.87 69 18 0.391 1.560.195 0.391 0.049 bad curve 1.11 85 1.06 55 0.56 33

The term “heteroatom” as used herein generally means any atom other thancarbon, hydrogen or oxygen. Preferred heteroatoms include oxygen (O),phosphorus (P), sulfur (S), nitrogen (N), silicon (S), arsenic (As),selenium (Se), and halogens, and preferred heteroatom functional groupsare haloformyl, hydroxyl, aldehyde, amine, azo, carboxyl, cyanyl,thocyanyl, carbonyl, halo, hydroperoxyl, imine, aldimine, isocyanide,iscyante, nitrate, nitrile, nitrite, nitro, nitroso, phosphate,phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which is fully saturated,having the number of carbon atoms designated (i.e. C1-C8 means one toeight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

The term “alkenyl”, by itself or as part of another substituent, means astraight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be mono- or polyunsaturated, having thenumber of carbon atoms designated (i.e. C2-C8 means two to eightcarbons) and one or more double bonds. Examples of alkenyl groupsinclude vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomersthereof.

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, or combination thereof,which may be mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e. C2-C8 means two to eight carbons) and one or moretriple bonds. Examples of alkynyl groups include ethynyl, 1- and3-propynyl, 3-butynyl and higher homologs and isomers thereof.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from alkyl, as exemplified by—CH₂—CH₂—CH₂—CH₂—. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S may be placed atany interior position of the heteroalkyl group. The heteroatom Si may beplaced at any position of the heteroalkyl group, including the positionat which the alkyl group is attached to the remainder of the molecule.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH3)-CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified by —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Accordingly, acycloalkyl group has the number of carbon atoms designated (i.e., C3-C8means three to eight carbons) and may also have one or two double bonds.A heterocycloalkyl group consists of the number of carbon atomsdesignated and from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. Additionally, for heterocycloalkyl, a heteroatom can occupythe position at which the heterocycle is attached to the remainder ofthe molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” and “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include alkyl substituted with halogen atoms, which can be thesame or different, in a number ranging from one to (2m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2m′+1) halogen atoms, where m′ is thetotal number of carbon atoms in the alkyl group). The term“perhaloalkyl” means, unless otherwise stated, alkyl substituted with(2m′+1) halogen atoms, where m′ is the total number of carbon atoms inthe alkyl group. For example the term “perhalo(C1-C4)alkyl” is meant toinclude trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl and the like.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.

The term “heteroaryl,” refers to aryl groups (or rings) that containfrom zero to four heteroatoms selected from N, O, and S, wherein thenitrogen and sulfur atoms are optionally oxidized and the nitrogenheteroatom are optionally quaternized. A heteroaryl group can beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of heteroaryl groups include 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR, halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR′—SO₂NR′″, —NR″CO₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′,—S(O)R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂, in a number ranging fromzero to three, with those groups having zero, one or two substituentsbeing particularly preferred. R′, R″ and R′″ each independently refer tohydrogen, unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstitutedaryl, aryl substituted with one to three halogens, unsubstituted alkyl,alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R′ and R″are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6- or 7-membered ring. For example, —NR′R″is meant to include 1-pyrrolidinyl and 4-morpholinyl. Typically, analkyl or heteroalkyl group will have from zero to three substituents,with those groups having two or fewer substituents being preferred inthe invention. More preferably, an alkyl or heteroalkyl radical will beunsubstituted or monosubstituted. Most preferably, an alkyl orheteroalkyl radical will be unsubstituted. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups such as trihaloalkyl (e.g., —CF₃ and—CH₂CF₃).

Preferred substituents for the alkyl and heteroalkyl radicals areselected from: —OR, ═O, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′, —NR′—SO₂NR″R′″,—S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂, where R′ and R″ areas defined above. Further preferred substituents are selected from:—OR′, ═O, —NR′R halogen, —OC(O)R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR″CO₂R′, —NR′—SO₂NR″R′″, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—C(O)NR″R′″, —NR′—SO₂NR″R′″, —NH—C(NH2)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R, —N₃, —CH(Ph)₂,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstitutedaryl)oxy-(C1-C4)alkyl. When the aryl group is1,2,3,4-tetrahydronaphthalene, it may be substituted with a substitutedor unsubstituted (C3-C7)spirocycloalkyl group. The(C3-C7)spirocycloalkyl group may be substituted in the same manner asdefined herein for “cycloalkyl”. Typically, an aryl or heteroaryl groupwill have from zero to three substituents, with those groups having twoor fewer substituents being preferred in the invention. In oneembodiment of the invention, an aryl or heteroaryl group will beunsubstituted or monosubstituted. In another embodiment, an aryl orheteroaryl group will be unsubstituted.

Preferred substituents for aryl and heteroaryl groups are selected from:halogen, —OR, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′, —CONR′R″,—C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R,—N₃, —CH(Ph)₂, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, whereR′ and R″ are as defined above. Further preferred substituents areselected from: halogen, —OR′, —OC(O)R′, —NR′R″, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —NR″C(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl.

The substituent —CO₂H, as used herein, includes bioisostericreplacements therefor; see, e.g., The Practice of Medicinal Chemistry;Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)q-U-, wherein T and U are independently —NH—, —O—, —CH₂— ora single bond, and q is an integer of from 0 to 2. Alternatively, two ofthe substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B-,wherein A and B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH₂)s-X—(CH₂)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)₂—, or —S(O)₂NR′—. The substituent R in —NR′— and —S(O)₂NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl. The term“pharmaceutically acceptable salts” is meant to include salts of theactive compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds of the invention contain relativelyacidic functionalities, base addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like. Certain specificcompounds of the invention contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the invention.

In addition to salt forms, the compounds may be in a prodrug form.Prodrugs of the compounds are those compounds that undergo chemicalchanges under physiological conditions to provide the compounds of theinvention. Additionally, prodrugs can be converted to the compounds ofthe invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the invention when placed in a transdermal patch reservoirwith a suitable enzyme or chemical reagent. Prodrugs are often usefulbecause, in some situations, they may be easier to administer than theparent drug. They may, for instance, be more bioavailable by oraladministration than the parent drug. The prodrug may also have improvedsolubility in pharmacological compositions over the parent drug. A widevariety of prodrug derivatives are known in the art, such as those thatrely on hydrolytic cleavage or oxidative activation of the prodrug. Anexample, without limitation, of a prodrug would be a compound of theinvention which is administered as an ester (the “prodrug”), but then ismetabolically hydrolyzed to the carboxylic acid, the active entity.

Subject compounds can exist in unsolvated forms as well as solvatedforms, including hydrated forms. In general, the solvated forms areequivalent to unsolvated forms and are intended to be encompassed withinthe scope of the invention. Subject compounds may exist in multiplecrystalline or amorphous forms. In general, all physical forms areequivalent for the uses contemplated and are intended to be within thescope of the invention.

Certain subject compounds possess asymmetric carbon atoms (opticalcenters) or double bonds; the racemates, diastereomers, geometricisomers and individual isomers are all intended to be encompassed withinthe scope of the invention.

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, losenges or the like in the case of solidcompositions.

A wide variety of suitable formulations and delivery systems, includingsuitable excipients or carriers and methods for preparing administrablecompositions, are known or apparent to those skilled in the art and aredescribed in more detail in such publications as Remington: The Scienceand Practice of Pharmacy (Pharmaceutical Press (2012). For example, inparticular embodiments the compositions are formulated or delivered inextended or controlled delivery systems, such as diffusion systems (e.g.reservoir devices, matrix devices, diffusion-controlled implants andtransdermal patches) and encapsulated and matrix dissolution systems,erosion products, osmotic pump systems, ion exchange resins, etc.

In particular embodiment the amount administered is far in excess ofthat (200 mg) currently indicated for Parkinson's Disease, and willpreferably be 0.5-10, 0.5-5, 0.5-2.5, 1-10, 1-5, 1-2.5, 2-10, or 2-5g/day, in unit dosage forms of 0.25, 0.5, 1, 1.5, 2 or 2.5 g. Thecompounds can be administered by a variety of methods including, but notlimited to, parenteral, topical, oral, or local administration, such asby aerosol or transdermally, for prophylactic and/or therapeutictreatment. Also, in accordance with the knowledge of the skilledclinician, the therapeutic protocols (e.g., dosage amounts and times ofadministration) can be varied in view of the observed effects of theadministered therapeutic agents on the patient, and in view of theobserved responses of the disease to the administered therapeuticagents.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including citations therein, are herebyincorporated by reference in their entirety for all purposes.

Examples: Discovery and Optimization of Benzotriazine Di-N-OxidesTargeting Replicating and Non-Replicating Mycobacterium tuberculosis

Survey of Bioreductively-activated Antimicrobial Scaffolds. Four classesof bioreductively-activated compounds (i.e. compounds having cellularactivities requiring the enzymatic transfer of electrons from reductaseenzymes) were selected for screening against Mtb H37Rv. The compoundsincluded 2- and 5-nitroimidazoles, 2-nitrofurans,quinoxaline-1,4-di-N-oxides, and BTOs (Table A). These particularclasses of compounds were selected based on prior reports of knownantimicrobial activities and based on availability through commercialsources or the National Cancer Institute (NCI) DevelopmentalTherapeutics Program compound repository.⁸⁻¹⁰ We screened the compoundsfor their MIC values against Mtb H37Rv strains from two sources, as wellas for their MIC values against NRP Mtb in the low-oxygen recovery assay(LORA)¹¹ (Table 1). The primary screening was performed in twoindependent laboratories with different sources of the H37Rv strain tovalidate that the activities were not only specific for a givenlaboratory strain, since wide variations in lab strains of H37Rv havebeen reported.¹² Additionally, we tested all of the compounds forcytotoxicity against Vero cells to give a selectivity index (SI)describing the relative cytotoxicities of compounds for Mtb over amammalian cell line.

TABLE A Chemical structures of bioreductively-activated compounds withknown antimicrobial activities. Nitroimidazoles

1 Nitrofurans

2 Quinoxaline Di-N-Oxides

3

4

5

6 Benzotriazine Di-N-Oxides

7

8a

TABLE 1 Antitubercular activity of different scaffolds ofbioreductively-activated antimicrobial compounds. TB MIC - TB MIC - LORAMIC - Vero Tox LUMO SRI ITR ITR CC₅₀ SI Classification Compound (eV)(μg/mL) (μg/mL) (μg/mL) (μg/mL) (CC₅₀/MIC) Isoniazid INH — 0.0600.091 >20 >50 >830 Rifampin RIF — 0.003 0.041 2.0 >50 >15000Nitroimidazole Metronidazole −1.303 >10 >32 11 >16 NA Etanidazole−1.644 >10 >32 26 >16 NA 1 −1.802 10 29 3.3 16 1.6 NitrofuransNitrofurantoin −1.801^(a) 16 28 32 23 1.1 Furazolidone −1.652^(a) 31 306.5 19 0.60 2 −1.660^(a) 0.98 3.8 3.7 >50 >50 Quinoxaline-1,4- 3 −1.45210 28 9.4 >16 >1.6 di-N-oxides 4 −2.436 >20 >32 >32 >16 NA 5 −1.919 1027 >32 >16 >1.6 6 −1.279 >10 >32 >32 >16 NA Benzotriazine- TPZ −1.6225.0 3.7 7.3 8.0 1.6 1,4-di-N-oxides 7 −1.703 5.0 3.7 0.97 38 7.6 8a−1.442 1.2 0.57 0.37 17 17 ^(a)NA = not available; ITR = Institute forTuberculosis Research, University of Illinois at Chicago.^(b)Calculation performed for Z stereochemistry around double bond.

The data from the survey of bioreductively-activated scaffolds showedthat the BTOs tested had consistent activities against both activelyreplicating and non-replicating Mtb. The MIC values ranged from 0.57-5μg/mL against the actively replicating H37Rv strains and from 0.37-7.3μg/mL in the LORA model of NRP. All of the compounds had somecytotoxicity against the Vero cells with moderate SI values. Using theresults of the initial survey, we selected the anti-TB activities of theBTOs, particularly analogs of 8a, for SAR development and optimizationstudies.

Synthesis and Activity of N-Substituted BTOs.

The first set of new BTOs synthesized contained analogs with diversesubstitutions at the 3-amine position of the ring (Scheme 1). Thesubstitutions at this position were designed to determine how steric andelectronic variations at this position affected the potency andselectivity of the compounds. The side chain modified analogs weresynthesized by reacting the primary amine starting materials (Table B)with 3-chloro-benzotriazine-1-N-oxide (9). The amine adducts were thenoxidized using pertrifluoroacetic acid, prepared in situ by addingtrifluoroacetic anhydride (TFAA) to a stirring solution of the compoundin excess hydrogen peroxide. The complete oxidation took 3-4 d withadditional equivalents of TFAA added until the oxidation was complete byTLC.

Scheme 1.

Synthesis of N-alkyl 1,2,4-benzotriazine-di-N-oxides. Reagents andconditions: (a) RNH₂ (2 eq.), TEA (2 eq.), DCM, 18 h, rt. (b) TFAA,H₂O₂, THF, 4 d.

TABLE B Amine diversity elements used for side chains on benzotriazines.

a

b

c

d

e

f

g

h

i

j

k

l

m

n

Results revealed that acyclic and cyclic alkyl groups of various sizeswere tolerated at the 3-amine position, but that none of themodifications significantly increased the compounds' SIs. (Table 2) Allof the compounds were active (MIC≦10 μg/mL); the most active were thosewith very hydrophobic side chains (8j and 8m). Incorporation of a polarether group (8i) into the side chain resulted in a significant loss ofactivity, but with a comparable reduction in cytotoxicity. The overallresults from this set indicated that both cyclic and acyclicsubstituents at this position were tolerated with the more hydrophobicgroups resulting in the most potent compounds. None of the modificationsin this set significantly improve the compounds' SIs, which ranged from2.9-8.3.

TABLE 2 Antitubercular activity of side-chain-substituted BTOs. TB TBLORA Vero MIC - MIC - MIC - Tox SI Com- LUMO SRI ITR ITR CC₅₀ (CC₅₀/pound (eV) (μg/mL) (μg/mL) (μg/mL) (μg/mL) MIC) 8b −1.652 2.5 1.8 1.8 218.3 8c −1.414 1.2 1.9 0.82 8.0 6.4 8d −1.589 1.2 0.94 0.84 8.9 7.1 8e−1.555 2.5 3.6 0.68 10 4.2 8f −1.536 2.5 3.8 0.75 8.9 3.6 8g −1.514 2.51.6 0.50 7.9 3.2 8h −1.530 5.0 1.9 0.99 26 5.3 8i −1.412 10.0 6.64.9 >50 >5.0 8j −1.523 0.62 0.22 <0.12 2.3 3.7 8k −1.495 1.2 0.92 0.368.9 7.1 8l −1.532 1.2 0.52 0.38 4.1 3.3 8m −1.553 0.62 0.60 0.42 3.4 5.58n −1.501 2.5 1.9 2.3 7.3 2.9

Synthesis and Activity of Ring-Substituted BTOs.

The mechanism of action for these BTOs is believed to be thebioreductive activation of the ring into a cytotoxic radical speciesthat causes irreparable DNA damage to Mtb. Given this mechanism, theelectron reduction potentials of the compounds should dictate theselectivity of the compounds for cytotoxicity to Mtb over mammaliancells. Variations to substitutions on the heterocyclic ring shouldsignificantly alter the electron reduction potentials and affect theselectivity index of the compounds.

The synthetic route for making the ring-modified BTOs began with eithera substituted aniline starting material (11) or a substituted2-nitroaniline (12) when available. (Scheme 2) The anilines wereacylated, nitrated with fuming nitric acid, and then deprotected byrefluxing in hydrochloric acid to give a set of 2-nitroanilines (12).¹³The 2-nitroanilines were then condensed with cyanamide in concentratedhydrochloric acid followed by treatment with base to form the1,2,4-benzotriazine-(1N)-oxide ring. The 3-aminobenzotriazines were thenconverted to 3-hydroxybenzotriazines by reaction with either sodiumnitrite or ten-butyl nitrite to form a diazonium intermediate, which washydrolyzed with acid. The intermediates were chlorinated by refluxing inphosphorus oxychloride to make the 3-chlorobenzotriazine-(1N)-oxideintermediates (14a-g). Intermediates 14a-g were then reacted with asubset of the primary amine side chains (Table B; side chains a, b, d,e, f, i and m). Side chains a, b, d and f were chosen because these lowmolecular weight side chains yielded potent compounds without addingunnecessary additional lipophilicity.¹⁴ Side chain i was chosen tosample a polar group at this position and m was chosen because ityielded one of the most potent compounds from the series ofside-chain-substituted analogs. This set of ring-substituted BTOs wasthen oxidized as described in Scheme 2 to give the compounds listed inTable 3.

All of the ring-substituted BTOs were tested for antitubercular activityand cytotoxicity (Table 3). The modifications to the ring substitutionsresulted in a wide range of potencies (0.15-5 μg/mL) and cytotoxicities(<2.5-100 μg/mL). Most importantly, several of the ring substitutionsled to a significant increase in SI values. The general trend observedwas that electron-donating substitutions led to reduced cytotoxicitiesagainst mammalian cells. That trend fits with the hypothesis thatlowering the one-electron reduction potential (E_(1/2)) of the compoundsmay be effective in increasing SI values.¹⁵ The best compounds from thisseries were those with small alkyl substitutions to the benzotriazinering, such as the 5-methyl and 6,7-cyclopentyl substitutions. These ringsystems yielded compounds with SIs near 50.

TABLE 3 Antitubercular activity of ring-substituted BTOs. TB TB LORAVero MIC - MIC - MIC - Tox SI Com- LUMO SRI ITR ITR CC₅₀ (CC₅₀/ pound(eV) (μg/mL) (μg/mL) (μg/mL) (μg/mL) MIC) 15aa −1.704 0.62 0.35 0.75 4.67.3 15ab −1.918 0.31 0.93 3.5 1.3 2.2 15ad −1.857 0.31 0.40 0.88 1.6 1015ae −1.820 0.31 0.33 0.34 2.3 7.3 15af −1.696 0.62 0.68 0.47 <2.5 <4.015ai −1.716 5.0 2.0 1.9 6.3 1.3 15am −1.808 0.31 <0.12 0.21 <2.5 <4.015ba −1.666 1.2 <0.12 0.41 2.6 2.1 15bb −1.870 0.62 0.21 0.22 1.3 2.215bd −1.812 0.15 <0.12 <0.12 1.6 10 15bf −1.655 0.62 0.94 0.63 2.6 4.215bi −1.675 1.2 1.4 0.78 2.6 2.1 15bm −1.759 0.15 0.22 0.23 1.1 7.3 15ca−1.652 0.62 0.49 0.46 4.2 6.7 15cd −1.793 0.15 0.46 0.41 3.8 24 15ce−1.757 0.15 0.35 0.46 18 110 15cm −1.749 0.31 0.36 0.41 0.80 2.6 15da−1.541 2.5 15 16 16 6.3 15dd −1.66 0.62 0.96 0.90 9.8 16 15dm −1.6290.62 0.97 1.6 >50 >80 15ea −1.409 0.62 0.51 0.48 22 36 15ed −1.567 0.310.47 0.43 22 70 15ee −1.524 0.31 0.43 0.27 3.7 12 15fa −1.254 1.2 0.950.83 36 29 15fd −1.352 0.62 0.96 0.44 34 54 15fm −1.244 1.2 0.930.36 >50 >41 15ga −1.195 2.5 3.5 2.5 >100 >40 15gd −1.324 2.5 5.12.9 >50 >20 15gm −1.284 5.0 5.6 3.5 >50 >10

Synthesis and Activity of Di-N-Alkyl BTOs.

The SAR data from the side chain and ring-substituted compoundssuggested that increasing the electron-donating groups around thebenzotriazine ring increased the selectivity index and that modifyingthe alkylamine side chains increased potency, but did not considerablyaffect the SI. From these observations, we concluded it was desirable tosynthesize new compounds with more electron-donating groups on thealkylamine side chain. In previous attempts, compounds withtri-substituted amine side chains could not be synthesized because thefinal oxidation reaction conditions did not produce the desireddi-N-oxide product. To make these compounds, we thus explored severalalternative oxidizing agents to pertrifluoroacetic acid, identifyingHOF.CH₃CN as a sufficiently strong reagent to carry out the desiredtransformation, without producing significant overoxidation sideproducts.^(16,17) This new route allowed synthesis of a series ofdi-N-alky BTOs (Scheme 3). For this route, it proved preferable tooxidize the 3-chlorobenzotriazine intermediates so that the aminediversification step was the last step in the library synthesis fromcommon intermediates 16 and 19.

When tested for antitubercular activity and cytotoxicity, the newdi-N-alkyl BTOs were found to have increased SIs (Table 4). Theantitubercular activities of the compounds were generally comparable tothose of the ring-substituted analogs, but cytotoxicities against Verocells were significantly reduced. From this set of compounds, weidentified several new compounds with SIs>50. From the set of secondaryamine starting materials used for this set of compounds (Scheme 3),small alkyl groups were found to have the greatest activities. Theaddition of polar or hydrophilic groups designed to increase solubilityresulted in a significant loss of anti-TB activity.

TABLE 4 Antitubercular activity of di-N-alkyl BTOs. TB TB LORA VeroMIC - MIC - MIC - Tox SI Com- LUMO SRI ITR ITR CC₅₀ (CC₅₀/ pound (eV)(μg/mL) (μg/mL) (μg/mL) (μg/mL) MIC) 17o −1.483 0.31 0.46 1.3 12 40 17p−1.624 2.5 3.7 19 5.4 2.2 17q −1.286 0.31 NT NT 17 56 20q −1.196 0.310.46 0.99 25 80 20r −1.311 0.62 0.40 0.49 21 13 20s −1.335 0.62 0.30 1.524 13 20t −1.344 0.31 0.15 0.41 17 53 20u −1.289 0.31 0.24 0.44 40 13020v −1.751 5.0 15 30 17 3.4 20w −1.293 1.2 0.66 0.47 >50 >40 20x −1.2585.0 13 15 >50 >10

Activity Against Drug-Resistant Mtb Strains.

We also tested a set of the most potent and selective BTOs against apanel of single-drug resistant Mtb strains (Table 5) to determinewhether or not the compounds had cross-resistance with existing TB drugsand maintained their potencies against diverse strains of Mtb. Theresulting data indicated that no cross-resistance occurred and that thecompounds had either equal or increased potencies against thedrug-resistant strains of Mtb.

TABLE 5 MIC of BTO's against Single Drug Resistant Mtb strains. MICValues (μg/mL) of Mtb Strains Resistant to: P-aminosalicyclicStreptomycin acid Isoniazid Kanamycin Ethionamide Ethambutol CompoundH37Rv ATCC 35820 ATCC 35821 ATCC 35822 ATCC 35827 ATCC 35830 ATCC 35837 8a 1.2 1.2 1.2 1.2 1.2 1.2 1.2  8c 2.5 1.2 1.2 2.5 2.5 1.2 2.5 15ea0.62 0.16 0.31 0.62 0.62 0.31 0.62 15fa 1.2 0.63 1.2 2.5 1.2 1.2 1.215fd 0.62 0.31 0.62 1.2 1.2 0.62 0.62 15ga 1.2 1.2 1.2 1.2 1.2 1.2 1.217q 0.31 0.02 0.62 0.31 0.31 0.31 0.31 20q 0.62 0.16 0.31 0.31 0.63 0.620.62 20r 0.62 0.15 0.31 0.15 0.31 0.31 0.31 20s 0.62 <0.07 0.15 <0.070.31 0.15 0.31 20t 0.31 <0.07 0.07 <0.07 0.15 0.07 0.15 20u 0.31 <0.070.15 0.07 0.15 0.15 0.31 20w 1.2 0.31 0.62 0.31 0.62 0.31 0.62

Antimicrobial Spectrum of Activity for BTOs.

Given the long treatment times and complex drug combination regimens,antitubercular drugs should have a narrow spectrum of antimicrobialactivity. Two BTOs, 8a and 20q, were profiled for their antimicrobialactivities against a diverse panel of Gram-positive and Gram-negativebacterial pathogens (SI Table 1). Remarkably, the BTOs were highlyselective in their activities for mycobacteria, with potent activityagainst Mtb and weaker activity against M. smegmatis. M. abscessus,which is naturally resistant to many classes of antibiotics, wasresistant to both 8a and 20q.¹⁸

Mutagenic Potential of BTOs.

A liability in developing any bioreductively-activated antimicrobialdrug is the potential for mutagenicity. Because the basis for activityof these compounds is the generation of intermediate radical species,undesirable mutagenicity poses a risk, as has been documented for manyclasses of bioreductive drugs, including the BTO tirapazamine(TPZ).^(19,20) The most convenient methodology for assessing thepotential to induce genetic damage is to use the plate incorporationmethod with Salmonella typhimurium strains TA98 and TA100, commonlyknown as the Ames assay. The Salmonella tester strains have mutations inthe histidine operon, a mutation that leads to defectivelipopolysaccharide (rfa), and a deletion that covers genes involved inthe synthesis of biotin (bio) and in the repair of ultraviolet-inducedDNA damage (uvrB).^(21,22) These mutations make the strains morepermeable to many molecules and increase their susceptibility to themutagenic effects of these molecules. Given that the hypothesizedmechanism of the BTO compounds is to selectively form cytotoxic radicalspecies in bacteria over mammalian cells, a microbial mutagenicity assayis not ideal, but was used to benchmark their activities in this assay.Two BTOs, 15fa and 20q, with good potency and selectivity profiles wereevaluated in the Ames assay, in both the presence and absence of anAroclor 1254-induced rat-liver metabolic activation system containing10% S9 (MA). Individual plate counts and their means and standarddeviations, along with the condition of the background lawn, arepresented in SI Table 2. There were dose-related increases in the numberof revertant colonies for both 15fa and 20q with both strains TA98 andTA100 in the presence and absence of metabolic activation. In order toassess their mutagenic potential in a non-microbial system 15fa and 20qwere also evaluated in the mouse lymphoma cell tk^(+/−)→tk^(−/−) genemutation (MOLY) assay—a routine genetic toxicology assay used to assessthe mutagenic potential of compounds in mammalian cells (Table6).^(23,24) In the presence or absence of metabolic activation (S9),15fa, at non-cytotoxic dose levels of 5, 10, 25, and 50 μg/mL showed asignificant increase in mutation frequency MF. For 20q, both in thepresence or absence of S9, mutation frequency did not increase atnon-cytotoxic dose levels up to 100 μg/mL for 4-hr exposure and up to 10μg/mL for 24-hr exposure in the absence of S9. In conclusion, 20q gavenegative response for mutation frequency in the presence or absence ofS9.

TABLE 6 Mutagenic potential of BTOs in MOLY assay. (S9 = Rat metabolicactivation; Positive = A significant induction of mutation frequency;Negative = No significant induction of mutation frequency; NT = Nottested) +S9, Dose −S9, 4-Hr −S9, 24-Hr +S9, 4-Hr 24-Hr Compound (μg/mL)Exposure Exposure Exposure Exposure 15fa 10 Positive NA PositivePositive 25 Positive NA Positive Positive 50 Positive NA PositivePositive 100  Positive NA Positive Positive 20q 10 Negative NegativeNegative Negative 25 Negative Negative Negative^(a) Negative  50^(a)Negative Negative Negative Negative 100^(a ) Negative Negative NegativeNegative ^(a)Induction of mutation frequency but slightly higher than 40× 10⁻⁶ net over mean solvent control, biologically considered negative.

Physiochemical Properties and Mouse Pharmacokinetics.

In order to assess the potential of BTOs as orally bioavailabletherapeutics, physiochemical properties and ADME properties wereprofiled for 8a and 20q. The solubility of these two compounds wasmeasured in triplicate using the shake-flask method in a 0.9% salinesolution at pH 7.4. Both 8a and 20q had acceptable solubilities of 1.35mg/mL and 0.28 mg/mL, respectively.

In order to validate BTOs as a new lead series for the treatment of TB,we also evaluated their systemic exposure in the mouse after oraladministration. A single dose (100 mg/kg) of 8a and 20q wereadministered to mice in order to profile the pharmacokinetics for theBTOs (Table 7). Our targeted drug candidate profile is an orallyadministered therapeutic that requires a maximum of once-a-day dosing,and preferable 1-3 times a week dosing in order to be compatible withcurrent first line TB drugs in a fixed dose combination tablet.²⁵ Inorder to achieve this profile, a mouse elimination half-life between4-12 hours is being targeted in our development program. The eliminationhalf-life was for each compound was shorter than our ideal targetvalues, but the T_(max) values indicate that they are rapidly absorbedafter oral administration (FIG. 1). Both compounds resulted in goodexposure based on C_(max) and AUC values, indicating these compoundsshow promise as antitubercular drugs.

TABLE 7 Pharmacokinetic Parameters for 8a and 20q after OralAdministration to Female Mice. Com- Dose t_(1/2) T_(max) C_(max)AUC_(last) AUC_(inf) pound (mg/kg) (hr) (hr) (ng/ml) (hr · ng/ml) (hr ·ng/ml)  8a 100 1.5 0.08 9540 6578 6937 20q 100 1.9 0.25 9767 11219 11248

Our SAR indicates that hydrophobic side chains generally resulted inmore potent antitubercular compounds, which was expected becausecompounds must pass through the hydrophobic mycobacterial cell wall toexert their activity. Increasing hydrophobicity also seemed to increasecytotoxicity to mammalian cells, presumably through increased cellpermeability. Modifications to the substitutions on the BTO ringresulted in substantial changes in the selectivity of the compounds. Ingeneral, introduction of electron-withdrawing groups such as halogens,resulted in more potent compounds against Mtb (MICs of 0.15-0.31 μg/mL),but those compounds were also more cytotoxic to Vero cells.Electron-donating substitutions on the ring tended to slightly decreasepotency against Mtb (MICs of 0.31-1.2 μg/mL), but substantiallydecreased toxicity, leading to compounds with overall improvedselectivity profiles. To balance these two opposing SAR trends weselected compounds with fused tricyclic ring systems (15f), which hadthe most balanced potency and selectivity profiles.

The development of the oxidation chemistry to produce the di-N-alkyl BTOcompounds allowed us to make compounds with 2-4 fold increases inpotency against Mtb, while either maintaining or decreasing toxicityagainst Vero cells. All of the compounds made in this series had verygood potencies (MICs of 0.31-0.62 μg/mL), except for compounds with morehydrophilic side chains containing a morpholino (17p), a sulfone (20v),or a sulfamide (20x); all those compounds demonstrated significantreductions in antitubercular activities (MICs of 2.5-5 μg/mL). Overall,the SAR trends suggested that optimal compounds should haveelectron-rich ring systems (lower E_(1/2) values; higher LUMO energies)with hydrophobic side chains to afford compounds with maximal potenciesand selectivities.

Analysis of the physiochemical properties and mouse pharmacokinetic datafor 8a and 20q show that the class has good drug-like properties. Thesolubilities of the compounds are generally very good, most likely dueto the polar nature of the N-oxide groups on the benzotriazine rings.20q had no mutagenic activity at concentrations up to 100 μg/mL.

General Procedure for Amination/Oxidation. Method A.

A primary amine (2.2 mmol) starting material was added to a stirringsolution of 9 (200 mg, 1.1 mmol) dissolved in DCM (10 mL) withtriethylamine (5.5 mmol) at room temperature. The solution was stirredfor 24-48 hours until all of the 9 starting material was consumed asmonitored by TLC (5% MeOH/DCM mobile phase). The bulky amine side chainsrequired heating to 45° C. for complete conversion. The entire reactionwas evaporated under reduced pressure to give the crude products 10a-nwhich were directly subjected to oxidation conditions. The crudemono-N-oxides were dissolved in a mixture of THF (8 mL) and 50% H₂O₂ (4mL), then slowly treated with TFAA (3.3 mmol) while stirring at roomtemperature. The oxidation was monitored by TLC and additionalequivalents of TFAA (3.3 mmol) were added every 12 hours until thereactions were >90% complete or no additional product was being formed.Upon completion of the reaction, the reaction was quenched with 10 mL ofsaturated sodium bicarbonate and extracted three times with 10 mL ofchloroform. The combined organic layers were evaporated and purified bysemi-preparative reversed-phase HPLC.

General Procedure for 3-Aminobenzotriazine 1-Oxides. Method B.

In a 1 L 3-neck flask with mechanical stirring and a reflux condenserwas placed a 2-nitroaniline (12a-h, 171 mmol), cyanamide (1.37 mol, 8eq.) and Et₂O (30 mL). The mixture was heated to 100° C. for 1 hour,then cooled to 50-55° C. Using an addition funnel concentrated HCl (72mL) was added dropwise with mechanical stirring (CAUTION: Highlyexothermic reaction.) Once the addition is complete and there no furthergas evolution, the mixture was heated to 110° C. for 3 hours. Thereaction was then cooled to 50° C. and a NaOH solution (7.5 M, 160 mL)was slowly added. This was then reheated to 110° C. with an oil bath for3 hours, cooled and poured into 3 L of H₂O. The resulting solid wasfiltered off, washed with H₂O and Et₂O, then dried under vacuum. Ifnecessary, the product was purified by flash chromatography using silicagel with a mobile phase gradient of 0-10% MeOH in CH₂Cl₂.

General Procedure for 3-Chlorobenzotriazine 1-Oxides. Method C.

NaNO₂ (1.5 g, 21 mmol) was added in small portions to a stirringsolution of a 3-aminobenzotriazine 1-oxide (13a-e, 7 mmol) intrifluoroacetic acid (15 mL) at room temperature. After 1 hour, 50 mL ofH₂O was added and the solid was filtered off and dried under vacuum. Thesolid was then suspended in 20 mL of POCl₃ and heated to reflux for 3hours. The solution was cooled, the majority of the excess POCl₃ wasremoved by distillation and then was dissolved in CH₂Cl₂ (40 mL). Thissolution was then washed with H₂O and brine, and the solvent wasevaporated. The crude residue was purified by flash chromatography usingsilica gel and a gradient of 0-10% EtOAc in CH₂Cl₂.

General Procedure for 3-Chlorobenzotriazine 1-Oxides. Method D.

tBuONO (90%, 15 mL) and H₂SO₄ (1 mL) were slowly added to a stirringsolution of a 3-aminobenzotriazine 1-oxide (13f-g, 9 mmol) in tBuOH (50mL) and H₂O (5 mL). After heating to 60° C. overnight the mixture addedto 100 g of ice and the resulting solid was filtered off and dried undervacuum. The solid was then suspended in 20 mL of POCl₃ and heated toreflux for 3 hours. The solution was cooled, the majority of the excessPOCl₃ was removed by distillation and then was dissolved in CH₂Cl₂ (40mL). This solution was then washed with H₂O and brine, and the solventwas evaporated. The crude residue was purified by flash chromatographyusing silica gel and a gradient of 0-10% EtOAc in CH₂Cl₂.

General Procedure for HOF.CH₃CN Oxidation. Method E.

(CAUTION: F₂ and HOF.CH₃CN are extremely strong oxidants and corrosivematerials) 10% F₂ with nitrogen was slowly bubbled into 100 mL of CH₃CNwith 10 mL of H₂O for 1 hour at −15° C. The resulting oxidant was addedin one portion to a cooled (0° C.) solution of the benzotriazine1-N-oxide (12.6 mmol) dissolved in CH₂Cl₂ (80 mL). The mixture wasstirred for 10 minutes, then quenched with saturated NaHCO₃ (40 mL). Thebiphasic mixture was then diluted with H₂O (40 mL) and extracted withCHCl₃ (80 mL). The organic layer was then washed with H₂O, brine anddried over MgSO₄. Solvent was evaporated and the product was purified byflash chromatography using silica gel and a mobile phase gradient of0-10% EtOAc in CH₂Cl₂. Isolated yields were from 35-40% with theremainder of the mass being recovered starting material.

General Procedure for Amination of3-Chlorobenzotriazine-1,4-Di-N-Oxides. Method F.

To a stirring solution of the 3-chlorobenzotriazine-1,4-di-N-oxide (2mmol) in dimethoxyethane (12 mL) at 0° C. was added an amine (2.2 mmol)and triethylamine (2.4 mmol). The mixture was allowed to warm to roomtemperature and was stirred for 16 hours. After the reaction, solventwas evaporated and the crude product was purified by flashchromatography using silica gel and a mobile phase gradient of 0-10%MeOH in CH₂Cl₂. Typical yields were from 80-90%.

3-(ethylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8a)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.28 (br-s, 1H), 8.18 (d, J=8.4 Hz, 1H), 8.10 (d, J=8.5 Hz, 1H), 7.90(t, J=7.5 Hz, 1H), 7.55 (t, J=8.6 Hz, 1H), 3.43 (m, 2H), 1.18 (t, J=7.0Hz, 3H). MS (ESI+): m/z 207.0 ((M+H)⁺).

3-(cyclopropylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8b)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.41 (d, J=2.4 Hz, 1H), 8.20 (dd, J=8.8, 0.8 Hz, 1H), 8.10 (dd, J=8.8,0.8 Hz, 1H), 7.92 (m, 1H), 7.56 (m, 1H), 2.76 (m, 1H), 0.74 (m, 4H). MS(ESI+): m/z 219.0 ((M+H)⁺).

3-(tert-butylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8c)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.31 (d, J=8.8 Hz, 1H), 8.26 (d, J=8.8 Hz, 1H), 7.83 (t, J=7.0 Hz, 1H),7.45 (t, J=7.0 Hz, 1H), 7.19 (s, 1H), 1.55 (s, 9H). MS (ESI+): m/z 235.0((M+H)⁺).

3-(cyclobutylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8d)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.44 (d, J=8.0 Hz, 1H), 8.17 (dd, J=8.8, 0.8 Hz, 1H), 8.10 (dd, J=8.8,0.8 Hz, 1H), 7.91 (m, 1H), 7.54 (m, 1H), 4.33 (m, 1H), 2.24 (m, 4H),1.67 (m, 2H). MS (ESI+): m/z 233.0 ((M+H)+).

3-(cyclopentylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8e)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.18 (dt, J=8.8, 0.8 Hz, 1H), 8.10-8.06 (m, 2H), 7.92 (m, 1H), 7.54 (m,1H), 4.18 (m, 1H), 1.92 (m, 2H), 1.70 (m, 4H), 1.56 (m, 2H). MS (ESI+):m/z 247.0 ((M+H)+).

3-(cyclohexylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8f)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.16 (dd, J=8.8, 0.8 Hz, 1H), 8.08 (dt, J=8.8, 0.8 Hz, 1H), 7.95 (d,J=9.2 Hz, 1H), 7.89 (m, 1H), 7.52 (m, 1H), 3.71 (m, 1H), 1.85 (m, 2H),1.72 (m, 2H), 1.58 (m, 1H), 1.51-1.42 (m, 2H), 1.35-1.26 (m, 2H),1.20-1.09 (m, 1H). MS (ESI+): m/z 261.0 ((M+H)+).

3-(cycloheptylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8g)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.18 (dt, J=8.8, 0.8 Hz, 1H), 8.09 (dt, J=8.8, 0.8 Hz, 1H), 7.96 (d,J=8.4 Hz, 1H), 7.91 (m, 1H), 7.53 (m, 1H), 3.92 (m, 1H), 1.89 (m, 2H),1.76-1.42 (m, 10H). MS (ESI+): m/z 275.0 ((M+H)+).

3-(pentan-2-ylamino)benzo[e][1,2,4]triazine 1,4-dioxide (8h)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.16 (dt, J=8.8, 0.4 Hz, 1H), 8.07 (dt, J=8.8, 0.4 Hz, 1H), 8.00 (d,J=9.2 Hz, 1H), 7.90 (m, 1H), 7.52 (m, 1H), 3.97 (m, 1H), 1.65 (m, 1H),1.49 (m, 1H), 1.30 (m, 2H), 1.20 (d, J=6.4 Hz, 3H), 0.83 (t, J=7.2 Hz,3H). MS (ESI+): m/z 249.0 ((M+H)+).

3-((1-methoxybutan-2-yl)amino)benzo[e][1,2,4]triazine 1,4-dioxide (8i)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.18 (dt, J=8.8, 0.8 Hz, 1H), 8.09 (dt, J=8.8, 0.8 Hz, 1H), 7.92 (m,2H), 7.54 (m, 1H), 3.50 (dd, J=10, 6.4 Hz, 1H), 3.40 (dd, J=10, 5.2 Hz,2H), 3.23 (s, 3H), 1.62 (m, 2H), 0.87 (m, 3H). MS (ESI+): m/z 265.0((M+H)+).

3-((4-(tert-butyl)cyclohexyl)amino)benzo[e][1,2,4]triazine 1,4-dioxide(8j)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.17 (td, J=8.4, 0.8 Hz, 1H), 8.08 (m, 1H), 7.97 (d, J=9.2 Hz, 1H), 7.91(m, 1H), 7.57-7.50 (m, 1H), 4.06 (m, 1H), 1.94 (m, 2H), 1.75 (m, 1H),1.58 (m, 2H), 1.14 (m, 4H), 0.83 (d, J=3.2 Hz, 9H). MS (ESI+): m/z 317.1((M+H)+).

(R)-3-((1-cyclohexylethyl)amino)benzo[e][1,2,4]triazine 1,4-dioxide (8k)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.16 (dd, J=8.8, 0.8 Hz, 1H), 8.08 (dd, J=8.8, 0.8 Hz, 1H), 7.89 (m,2H), 7.51 (m, 1H), 3.75 (m, 1H), 1.72-1.55 (m, 6H), 1.18 (d, J=6.8 Hz,3H), 1.12 (m, 3H), 0.92 (m, 2H). MS (ESI+): m/z 289.0 ((M+H)+).

3-((5-methylhexan-2-yl)amino)benzo[e][1,2,4]triazine 1,4-dioxide (8l)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.16 (dd, J=8.8, 0.8 Hz, 1H), 8.09 (dd, J=8.4, 0.4 Hz, 1H), 7.94 (d,J=9.6 Hz, 1H), 7.89 (m, 1H), 7.52 (m, 1H), 3.91 (m, 1H), 1.66 (m, 1H),1.50 (m, 2H), 1.20 (d, J=6.8 Hz, 3H), 1.15 (m, 2H), 0.83 (dd, J=6.4, 2.0Hz, 6H). MS (ESI+): m/z 277.0 ((M+H)+).

3-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)benzo[e][1,2,4]triazine1,4-dioxide (8m)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.18 (dt, J=8.8, 0.4 Hz, 1H), 8.09 (dd, J=8.8, 0.8 Hz, 1H), 7.90 (m,1H), 7.73 (d, J=6.8 Hz, 1H), 7.53 (m, 1H), 3.65 (m, 1H), 2.26 (m, 2H),1.69 (m, 2H), 1.50 (m, 3H), 1.14 (m, 3H). MS (ESI+): m/z 273.0 ((M+H)+).

3-((1R,3S,5r,7r)-adamantan-2-ylamino)benzo[e][1,2,4]triazine 1,4-dioxide(8n)

Synthesized from 9 by Method A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.18 (dd, J=8.8, 0.8 Hz, 1H), 8.11 (dd, J=8.8, 1.2 Hz, 1H), 7.93 (m,1H), 7.55 (m, 1H), 7.39 (d, J=8.4 Hz, 1H), 4.00 (m, 1H), 2.04 (m, 2H),1.88 (m, 8H), 1.72 (br s, 2H), 1.61 (m, 2H). MS (ESI+): m/z 313.1((M+H)+).

3-(ethylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-dioxide (15aa)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (CDCl₃, 400 MHz): 8.26 (dd, J=4.9, 9.6; 1H), 7.92 (dd,J=1.7, 8.0; 1H), 7.57 (m, 1H), 6.94 (br-t, 1H), 3.57 (p, J=7.3, 2H),1.29 (t, J=7.3, 3H). MS (ESI+): m/z 224.9 ((M+H)+).

3-(cyclopropylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-dioxide (15ab)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.37 (br s, 1H), 8.15 (dd,J=9.6, 5.2 Hz, 1H), 7.97 (dd, J=8.4, 2.8 Hz, 1H), 7.84 (m, 1H), 2.72 (m,1H), 0.72 (m, 4H). MS (ESI+): m/z 236.9 ((M+H)+).

3-(cyclobutylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-dioxide (15ad)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.43 (d, J=8.0 Hz, 1H), 8.15(dd, J=9.6, 5.2 Hz, 1H), 7.93 (dd, J=8.8, 2.8 Hz, 1H), 7.83 (m, 1H),4.30 (m, 1H), 2.23 (m, 4H), 1.67 (m, 2H). MS (ESI+): m/z 250.9 ((M+H)+).

3-(cyclopentylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-dioxide (15ae)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.14 (dd, J=9.6, 5.2 Hz, 1H),7.97 (m, 1H), 7.94 (dd, J=8.4, 2.8 Hz, 1H), 7.83 (m, 1H), 4.13 (m, 1H),1.91 (m, 2H), 1.67 (m, 4H), 1.55 (m, 2H). MS (ESI+): m/z 265.0 ((M+H)+).

7-fluoro-3-((1-methoxybutan-2-yl)amino)benzo[e][1,2,4]triazine1,4-dioxide (15ag)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.16 (dd, J=9.6, 5.2 Hz, 1H),7.95 (dd, J=9.2, 2.8 Hz, 1H), 7.85 (m, 2H), 3.95 (m, 1H), 3.49 (dd,J=10, 6.4 Hz, 1H), 3.39 (dd, J=9.6, 5.2 Hz, 1H), 3.23 (s, 3H), 1.61 (m,2H), 0.86 (t, J=7.2 Hz, 3H). MS (ESI+): m/z 283.0 ((M+H)+).

3-(cyclohexylamino)-7-fluorobenzo[e][1,2,4]triazine 1,4-dioxide (15aj)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.14 (dd, J=9.2, 5.2 Hz, 1H),7.95 (dd, J=8.8, 2.8 Hz, 1H), 7.91 (m, 1H), 7.83 (m, 1H), 3.69 (m, 1H),1.84 (m, 2H), 1.71 (m, 2H), 1.58 (m, 1H), 1.51-1.41 (m, 2H), 1.35-1.25(m, 2H), 1.12 (m, 1H). MS (ESI+): m/z 279.0 ((M+H)+).

3-(1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)-7-fluorobenzo[e][1,2,4]triazine1,4-dioxide (15an)

Synthesized from 4-fluoro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.15 (dd, J=9.6, 4.8 Hz, 1H),7.95 (dd, J=9.2, 2.8 Hz, 1H), 7.84 (m, 1H), 7.74 (d, J=7.2 Hz, 1H), 3.61(m, 1H), 2.28 (d, J=4.0 Hz, 1H), 2.22 (br s, 1H), 1.69 (m, 2H), 1.56 (m,1H), 1.45 (m, 2H), 1.12 (m, 3H). MS (ESI+): m/z 291.0 ((M+H)+).

7-chloro-3-(ethylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15ba)

Synthesized from 4-chloro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.41 (t, J=6.0 Hz, 1H), 8.19 (d,J=2.4 Hz, 1H), 8.10 (d, J=9.2 Hz, 1H), 7.91 (dd, J=9.2, 2.4 Hz, 1H),3.41 (m, 2H), 1.17 (t, J=7.2 Hz, 3H). MS (ESI+): m/z 241.0 ((M+H)+).

7-chloro-3-(cyclopropylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15bb)

Synthesized from 4-chloro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.47 (br s, 1H), 8.19 (d, J=2.0Hz, 1H), 8.09 (d, J=9.2 Hz, 1H), 7.90 (dd, J=9.2, 2.0 Hz, 1H), 2.73 (m,1H), 0.77-0.67 (m, 4H). MS (ESI+): m/z 253.0 ((M+H)+).

7-chloro-3-(cyclobutylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15bd)

Synthesized from 4-chloro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.55 (d, J=7.6 Hz, 1H), 8.16 (d,J=1.6 Hz, 1H), 8.09 (d, J=9.2 Hz, 1H), 7.89 (dd, J=9.2, 2.0 Hz, 1H),4.31 (m, 1H), 2.22 (m, 4H), 1.66 (m, 2H). MS (ESI+): m/z 267.0 ((M+H)+).

7-chloro-3-((1-methoxybutan-2-yl)amino)benzo[e][1,2,4]triazine1,4-dioxide (15bg)

Synthesized from 4-chloro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.18 (d, J=2.4 Hz, 1H), 8.10 (d,J=9.2 Hz, 1H), 7.96 (d, J=8.8 Hz, 1H), 7.90 (dd, J=9.2, 2.4 Hz, 1H),3.97 (m, 1H), 3.49 (dd, J=9.6, 6.4 Hz, 1H), 3.39 (m, 1H), 3.23 (m, 3H),1.61 (m, 2H), 0.86 (t, J=7.2 Hz, 3H). MS (ESI+): m/z 299.0 ((M+H)+).

7-chloro-3-(cyclohexylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15bj)

Synthesized from 4-chloro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.17 (d, J=2.4 Hz, 1H), 8.08 (d,J=9.2 Hz, 1H), 8.02 (d, J=8.8 Hz, 1H), 7.89 (dd, J=9.2, 2.4 Hz, 1H),3.70 (m, 1H), 1.82 (m, 2H), 1.70 (m, 2H), 1.57 (m, 1H), 1.46 (m, 2H),1.31 (m, 2H), 1.12 (m, 1H). MS (ESI+): m/z 295.1 ((M+H)+).

3-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)-7-chlorobenzo[e][1,2,4]triazine1,4-dioxide (15bn)

Synthesized from 4-chloro-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.18 (dd, J=2.4, 0.4 Hz, 1H),8.08 (dd, J=9.6, 0.8 Hz, 1H), 7.90 (dd, J=9.2, 2.4 Hz, 1H), 7.85 (m,1H), 3.62 (m, 1H), 2.28 (d, J=4.0 Hz, 1H), 2.22 (br s, 1H), 1.69 (m,2H), 1.56 (m, 1H), 1.47 (m, 2H), 1.14 (m, 3H). MS (ESI+): m/z 307.1((M+H)+).

7-bromo-3-(ethylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15ca)

Synthesized from 4-bromo-2-nitroaniline using Method C and Method A. Redsolid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.41 (t, J=6.4 Hz, 1H), 8.32 (m,1H), 8.01 (m, 2H), 3.40 (m, 2H), 1.17 (t, J=7.2 Hz, 3H). MS (ESI+): m/z286.9 ((M+H)+).

7-bromo-3-(cyclobutylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15cd)

from Synthesized from 4-bromo-2-nitroaniline using Method C and MethodA. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.58 (m, 1H), 8.31 (m, 1H),8.02 (m, 2H), 4.32 (m, 1H), 2.32 (m, 4H), 1.67 (m, 2H). MS (ESI+): m/z312.9 ((M+H)+).

7-bromo-3-(cyclopentylamino)benzo[e][1,2,4]triazine 1,4-dioxide (15ce)

Synthesized from 4-bromo-2-nitroaniline using Method C and Method A. Redsolid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.32 (t, J=1.2 Hz, 1H), 8.14 (d,J=8.0 Hz, 1H), 8.01 (m, 2H), 4.16 (m, 1H), 1.92 (m, 2H), 1.70 (m, 4H),1.56 (m, 2H). MS (ESI+): m/z 326.9 ((M+H)+).

3-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)-7-bromobenzo[e][1,2,4]triazine1,4-dioxide (15cn)

Synthesized from 4-bromo-2-nitroaniline using Method C and Method A. Redsolid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.33 (t, J=1.2 Hz, 1H), 8.02 (m,2H), 7.89 (m, 1H), 3.62 (m, 1H), 2.30 (m, 1H), 2.23 (m, 1H), 1.72 (m,2H), 1.59-1.40 (m, 3H), 1.23-1.01 (m, 3H). MS (ESI+): m/z 352.9((M+H)+).

3-(ethylamino)-7-methoxybenzo[e][1,2,4]triazine 1,4-dioxide (15da)

Synthesized from 4-methoxy-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.08 (t, J=6.0 Hz, 1H), 8.04 (d,J=9.6 Hz, 1H), 7.57 (dd, J=9.6, 2.8 Hz, 1H), 7.46 (d, J=2.4 Hz, 1H),3.90 (s, 3H), 3.38 (m, 2H), 1.17 (t, J=7.2 Hz, 3H). MS (ESI+): m/z 237.0((M+H)+).

3-(cyclobutylamino)-7-methoxybenzo[e][1,2,4]triazine 1,4-dioxide (15dd)

Synthesized from 4-methoxy-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.25 (d, J=8.0 Hz, 1H), 8.05 (d,J=9.6 Hz, 1H), 7.57 (dd, J=9.2, 2.8 Hz, 1H), 7.45 (d, J=2.4 Hz, 1H),4.30 (m, 1H), 3.90 (s, 3H), 2.22 (m, 4H), 1.67 (m, 2H). MS (ESI+): m/z263.0 ((M+H)+).

3-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)-7-methoxybenzo[e][1,2,4]triazine1,4-dioxide (15dn)

Synthesized from 4-methoxy-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.04 (d, J=9.6 Hz, 1H), 7.56 (m,2H), 7.46 (d, J=2.4 Hz, 1H), 3.90 (s, 3H), 3.61 (m, 1H), 2.30 (d, J=4.0Hz, 1H), 2.23 (br s, 1H), 1.69 (m, 2H), 1.57-1.35 (m, 3H), 1.23-1.09 (m,3H). MS (ESI+): m/z 303.0 ((M+H)+).

Ethyl-(5-methyl-1,4-dioxy-benzo[1,2,4]triazin-3-yl)-amine (15ea)

Synthesized from 6-methyl-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.34 (t, J=6.4 Hz, 1H), 8.05(dq, J=8.8, 0.8 Hz, 1H), 7.60 (m, 1H), 7.38 (m, 1H), 3.40 (p, J=6.8 Hz,2H), 2.95 (s, 3H), 1.18 (t, J=7.2 Hz, 3H). MS (ESI+): m/z 221.0((M+H)+).

3-(cyclobutylamino)-5-methylbenzo[e][1,2,4]triazine 1,4-dioxide (15ed)

Synthesized from 6-methyl-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.43 (d, J=8.0 Hz, 1H), 8.04 (d,J=8.8 Hz, 1H), 7.59 (dt, J=7.2, 1.2 Hz, 1H), 7.38 (m, 1H), 4.31 (m, 1H),2.96 (s, 3H), 2.27-2.17 (m, 4H), 1.68 (m, 2H). MS (ESI+): m/z 247.0((M+H)+).

3-(cyclopentylamino)-5-methylbenzo[e][1,2,4]triazine 1,4-dioxide (15ee)

Synthesized from 6-methyl-2-nitroaniline using Method C and Method A.Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.05 (m, 1H), 7.95 (d, J=7.6 Hz,1H), 7.59 (dt, J=7.2, 1.2 Hz, 1H), 7.38 (dd, J=8.8, 7.2 Hz, 1H), 4.15(m, 1H), 2.96 (s, 3H), 1.94 (m, 2H), 1.68 (m, 4H), 1.57 (m, 2H). MS(ESI+): m/z 261.0 ((M+H)+).

3-(ethylamino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide(15fa)

Synthesized from 6-nitro-2,3-dihydro-1H-inden-5-amine using Method D andMethod A. Red solid. ¹H-NMR (CDCl₃, 400 MHz): δ 8.11 (s, 1H), 8.07 (s,1H), 6.94 (br-t, 1H), 3.59 (p, J=7.2, 2H), 3.09 (t, J=6.7, 2H), 3.03 (t,J=7.7, 2H), 2.20 (p, J=7.5, 2H), 1.33 (t, J=7.3, 3H). MS (ESI+): m/z247.0 ((M+H)+).

Cyclobutyl-(5,8-dioxy-2,3-dihydro-1H-5,6,8-triaza-cyclopenta[b]naphthalen-7-yl)-amine(15fd)

Synthesized from 6-nitro-2,3-dihydro-1H-inden-5-amine using Method D andMethod A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.32 (d, J=8.0 Hz,1H), 7.99 (br s, 1H), 7.95 (br s, 1H), 4.32 (m, 1H), 3.07-2.97 (m, 4H),2.22 (m, 4H), 2.07 (m, 2H), 1.67 (m, 2H). MS (ESI+): m/z 273.0 ((M+H)+).

3-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (15fn)

Synthesized from 6-nitro-2,3-dihydro-1H-inden-5-amine using Method D andMethod A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.00 (br s, 1H), 7.94(br s, 1H), 7.63 (d, J=6.8 Hz, 1H), 3.63 (m, 1H), 3.07-2.97 (m, 4H),2.30 (d, J=4.0 Hz, 1H), 2.24 (br s, 1H), 2.07 (m, 2H), 1.70 (m, 2H),1.57 (m, 1H), 1.49 (m, 2H), 1.15 (m, 3H). MS (ESI+): m/z 313.0 ((M+H)+).

3-(ethylamino)-6-methoxy-7-methylbenzo[e][1,2,4]triazine 1,4-dioxide(15ga)

Synthesized from 5-methoxy-4-methyl-2-nitroaniline using Method D andMethod A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.17 (t, J=6.0 Hz,1H), 7.99 (d, J=1.2 Hz, 1H), 7.33 (s, 1H), 4.00 (s, 3H), 3.42-3.36 (m,2H), 2.26 (d, J=0.8 Hz, 3H), 1.17 (t, J=7.2 Hz, 3H). MS (ESI+): m/z251.0 ((M+H)+).

3-(cyclobutylamino)-6-methoxy-7-methylbenzo[e][1,2,4]triazine1,4-dioxide (15gd)

Synthesized from 5-methoxy-4-methyl-2-nitroaniline using Method D andMethod A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.31 (d, J=8.4 Hz,1H), 7.98 (m, 1H), 7.33 (s, 1H), 4.31 (m, 1H), 4.00 (s, 3H), 2.26 (d,J=0.8 Hz, 3H), 2.22 (m, 4H), 1.67 (m, 2H). MS (ESI+): m/z 277.0((M+H)+).

3-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-ylamino)-6-methoxy-7-methylbenzo[e][1,2,4]triazine1,4-dioxide (15gn)

Synthesized from 5-methoxy-4-methyl-2-nitroaniline using Method D andMethod A. Red solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.01 (d, J=1.2 Hz,1H), 7.60 (d, J=6.8 Hz, 1H), 7.32 (s, 1H), 4.00 (s, 3H), 3.63 (m, 1H),2.33 (m, 1H), 2.27 (s, 3H), 2.24 (br s, 1H), 1.69 (m, 2H), 1.49 (m, 3H),1.16 (m, 3H). MS (ESI+): m/z 317.0 ((M+H)+).

3-chlorobenzo[e][1,2,4]triazine 1,4-dioxide (16)

Synthesized from 9 by Method E. Yellow solid. ¹H-NMR (CDCl₃, 300 MHz): δ8.55 (d, J=8.5, 1H), 8.48 (d, J=8.6, 1H), 8.07 (t, J=8.5 Hz, 1H), 7.90(t, J=8.6 Hz, 1H). MS (ESI+): m/z 198.0 ((M+H)+).

3-chloro-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (19)

Synthesized from 18 by Method E. Yellow solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.34 (s, 1H), 8.26 (s, 1H), 3.17 (q, J=7.8 Hz, 4H), 2.27 (quin, J=7.8,2H). MS (ESI+): m/z 238.0 ((M+H)+).

3-(diethylamino)benzo[e][1,2,4]triazine 1,4-dioxide (17o)

Synthesized from 16 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.36 (d, J=7.4 Hz, 1H), 8.31 (d, J=7.4 Hz, 1H), 7.85 (t, J=7.4 Hz,1H), 7.54 (t, J=7.4 Hz, 1H), 3.81 (q, J=6.9 Hz, 4H), 1.32 (t, J=7.0 Hz,6H). (ESI+): m/z 235.0 ((M+H)+).

3-morpholinobenzo[e][1,2,4]triazine 1,4-dioxide (17p)

Synthesized from 16 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.39 (d, J=8.7 Hz, 1H), 8.35 (d, J=9.6 Hz, 1H), 7.90 (t, J=7.0 Hz,1H), 7.65 (t, J=7.0, 1H), 3.91 (m, 4H), 3.87 (m, 4H). (ESI+): 249.1((M+H)+).

3-(pyrrolidin-1-yl)benzo[e][1,2,4]triazine 1,4-dioxide (17q)

Synthesized from 16 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.31 (d, J=5.8 Hz, 2H), 7.84 (t, J=5.8, 1H), 7.49 (t, J=5.8 Hz, 1H),4.04 (m, 4H), 1.98 (m, 4H). (ESI+): m/z 233.0 ((M+H)+).

3-(pyrrolidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20q)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.11 (s, 1H), 8.10 (s, 1H), 4.03 (t, J=6.6, 4H), 3.07 (t, J=7.3 Hz,2H), 3.04 (t, J=7.5 Hz, 2H), 2.19 (quin, J=7.4 Hz, 2H), 1.98 (m, 4H). MS(ESI+): m/z 273.0 ((M+H)+).

3-(piperidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20r)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.15 (s, 1H), 8.11 (s, 1H), 3.76 (m, 4H), 3.05 (m, 4H), 2.18 (quintet,J=7.5 Hz, 2H), 1.69 (m, 6H). MS (ESI+): m/z 287.0 ((M+H)+).

3-(dimethylamino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20s)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.15 (s, 1H), 8.10 (s, 1H), 3.31 (s, 6H), 3.05 (quint, J=6.9 Hz, 4H),2.15 (quint, J=7.3 Hz, 2H). MS (ESI+): m/z 247.0 ((M+H)+).

3-(benzyl(methyl)amino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20t)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.24 (s, 1H), 8.16 (s, 1H), 7.25-7.35 (m, 5H), 5.1 (s, 2H), 3.17 (s,3H), 3.12 (quint, J=7.6 Hz, 4H), 2.21 (quint, J=7.6 Hz, 2H). MS (ESI+):m/z 323.0 ((M+H)+).

3-(methyl(phenethyl)amino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20u)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.09 (s, 1H), 8.05 (s, 1H), 7.21 (d, J=7.4 Hz, 2H), 7.11 (t, J=5.2 Hz,2H), 7.00 (t, J=7.4 Hz, 1H), 4.16 (t, J=7.6 Hz, 2H), 3.24 (s, 3H),2.97-3.11 (m, 6H), 2.18 (quint, J=7.6 Hz, 2H). MS (ESI+): m/z 337.0((M+H)+).

3-(thiomorpholinosulfone-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20v)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.17 (s, 1H), 8.14 (s, 1H), 4.35 (m, 2H), 3.26 (m, 2H), 3.12 (m, 2H),2.23 (quint, J=7.4 Hz, 2H). MS (ESI+): m/z 337.0 ((M+H)+).

3-(2-methylpiperidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20w)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.16 (s, 1H), 8.12 (s, 1H), 4.18 (t, J=7.0 Hz, 1H), 4.18 (d, J=13.2Hz, 1H), 3.32 (m, 1H), 3.03 (m, 4H), 2.16 (quint, J=7.3 Hz, 2H), 1.92(m, 1H), 1.62 (m, 6H), 1.33 (d, J=7.0 Hz, 3H). MS (ESI+): m/z 301.0((M+H)+).

3-(4-(methylsulfonyl)piperazin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20x)

Synthesized from 19 using Method F. Red solid. ¹H-NMR (CDCl₃, 300 MHz):δ 8.15 (s, 1H), 8.12 (s, 1H), 3.95 (m, 4H), 3.42 (m, 4H), 3.07 (quint,J=5.9 Hz, 4H), 2.79 (s, 3H), 2.17 (quint, J=7.7 Hz, 2H). MS (ESI+): m/z366.0 ((M+H)+).

Calculation of LUMO Energies:

Molecular orbital energy calculations were performed using thesemiempirical quantum chemistry program MOPAC2009.⁵² The calculation wasperformed with the Neglect of Diatomic Differential Overlap (NDDO)approximation method with the PM6 parameterization. The geometries ofall compounds were optimized using the default Eigenvector Followingroutine until a self-consistent field was achieved. The energies of theLUMO for each molecule is approximated by the eigenvalues (in eV) of theappropriate orbital generated using the EIGEN and VECTORS keywords.

Bacterial Strains, Growth Conditions, and Chemicals:

Mtb strains were obtained from the American Type Culture Collection(ATCC, VA) and Colorado State University (CSU) and cultured in rollerbottles (Corning Inc) using Middlebrook 7H9 broth (Difco Laboratories)supplemented with 0.2% glycerol, 0.05% Tween-80, and 10%albumin-dextrose-catalase (Difco Laboratories). Middlebrook 7H10 agar(Difco Laboratories), supplemented with 0.2% glycerol and 10% oleicacid-albumin-dextrose-catalase (Difco Laboratories), was used tovisualize colonies. Mtb H37Rv was grown in 7H9 broth at 37° C. to themid-log phase. The antibiotics and resazurin were purchased from Sigma(St. Louis, Mo.) and resuspended according to the manufacturer'sinstructions. Experiments performed at the Institute for TuberculosisResearch at the University of Illinois Chicago (TB MIC-ITR) wereperformed according to published methods.^(11,53)

Determination of Antibiotic Susceptibility:

To determine the MIC of compounds against Mtb, the resazurin-basedmicroplate assay was performed. Briefly, the compounds were resuspendedin DMSO and tested in a range from 10-0.08 mg/L following a two-folddilution scheme. After addition of the bacterial cells ˜10⁵ colonyforming unit (CFU)/mL, the 96 well plates were incubated at 37° C. for 5d. The addition of 0.05 mL of 0.1% resazurin followed, with additionalincubation for 2 d at 37° C. Fluorescence was measured using aFluoroskan Ascent or Victor 3 microplate fluorimeter (Thermo Scientific,USA) with an excitation of 530 nm and emission of 590 nm. Wellscontaining compounds only were used to detect autofluorescence ofcompounds. The lowest drug concentration that inhibited ≧90% growth wasconsidered to be the MIC. In addition to the fluorescence readouts, allof the MIC values are also scored by visual inspection for confirmationof the MIC value. A two-fold variation in MIC was considered to bewithin the error range of the assay. The final concentration of DMSO inall wells was 0.625%. These data are presented in Tables 1-5.

Antimicrobial Activity Against NRP Mtb Cells:

The antimicrobial activity of various compounds against NRP Mtb cellswas determined as described previously using LORA.⁷ Briefly, Mtb H37Rvcells were suspended in Middlebrook 7H12 broth and sonicated for 15 s.Cultures were diluted to obtain an OD₅₇₀ of 0.03-0.05 and 3000-7000relative light units (RLU) per 100 μL. Two-fold serial dilutions ofantimicrobial agents were prepared in a volume of 100 μL in black96-well microtiter plates, and 100 μL of the cell suspension was added.The microplate cultures were placed under anaerobic conditions(O₂<0.16%) using an Anoxomat Model WS-8080 (MART Microbiology) usingthree cycles of evacuation and filling with a mixture of 10% H₂, 5% CO₂,and the balance N₂. An anaerobic indicator strip was placed inside thechamber to visually confirm the removal of O₂. Plates are incubated at37° C. for 10 d and then transferred to an ambient gaseous condition (5%CO₂-enriched air) incubator for a 28-h “recovery”. On day 11 (after the28-h aerobic recovery), 100 μL culture were transferred to white 96-wellmicrotiter plates for determination of luminescence. The MIC was definedas the lowest drug concentration effecting an inhibition of ˜90%relative to drug-free controls.

Cytotoxicity Against Vero Cells.

The compounds were tested for cytotoxicity against Vero cells using theCell Titer-Glo Luminescent Cell Viability Assay (Promega). The Verocells (Vero ATCC CCL-81) were grown and maintained in minimal essentialmedium (MEM)+0.25% fetal bovine serum (FBS). Compound dilutions weredone in accordance with Clinical and Laboratory Standards Institute(CLSI) guidelines in 96-well plates. Each well contained 5 μL ofcompound and 95 μL of host Vero cells at a concentration of ˜5×10⁴cells/mL. Plates were incubated for 72 h at 37° C.+5% CO₂, followed byequilibration of the plate to room temperature for ˜30 min Addition ofthe Cell Titer-Glo reagent followed, with mixing the contents for 2 minon an orbital shaker to induce cell lysis and further incubation at roomtemperature for 10 min to stabilize the luminescent signal. The plateswere read and the luminescence was recorded. Each test plate contained aset of controls including media only (for background subtraction) and0.625% DMSO controls (for calculating the percent viability for all testwells). In addition, each test plate contained an ATP standard curve,which was used to calculate the ATP units in each test well. Thetoxicity was defined as the concentration at which host cells are 50%viable (IC₅₀).

Mouse Lymphoma Cell Tk+/−→Tk−/− Gene Mutation Assay.

Experiments were conducted using a standard procedure for evaluatingmutagenic potential.^(23,24) The details on cell line, experimentalprocedures and data interpretaion have been previously published.⁵⁴ Thefollowing endpoints were evaluated in cells following exposure to 15faand 20q: cell growth during expression periods, relative suspensiongrowth, relative total growth, relative cloning efficiency, mutationfrequency, and numbers of small (≦0.6 mm in diameter) colonies fromtrifluorothymidine-resistant (TFT^(r)) cells.

Solubility Assays.

Compounds were ground into a fine powders using a mortar and pestle andadded to a 25 mL glass Erlenmeyer flask with 5 mL of a 0.9% salinesolution at pH 7.4. Approximately 20 mg of compound was added to eachflask to ensure saturation. The flasks were then vigorously stirredusing a magnetic stir bar at 25° C. for 48 hours. The samples were thenfiltered through a Whatman PVDF membrane (0.45 μm pore size) syringefilter. The concentration of compound in the supernatant was thenmeasured using HPLC quantitation at the maximum absorption wavelengthfor each compound. The integration was then fit to a standard curvegenerated for each compound (R² values>0.99) to determine theequilibrium solubility for each compound.

Pharmacokinetic Studies in Female CD1 Mice.

All pharmacokinetic studies were performed in accordance with SRIInternational's animal care policies in an AAALAC and OLAW accreditedfacility. The procedure for the pharmacokinetics studies followed apreviously described method.⁵⁵ Briefly, the plasma pharmacokinetics ofselected BTO derivatives were determined in female CD1 mice afteradministration of a single dose (100 mg/kg) by oral gavage. Blood wascollected from three mice per time point at 5, 15, 30, and 60 min and 2,4, 6, 8, and 24 h after dose administration. Means and standarddeviations were calculated for the plasma drug concentrations at eachtime point. Pharmacokinetic analysis was performed using noncompartmental methods (WinNonlin® Professional, Version 5.2, PharsightCorp, Mountain View, Calif.). The following parameters were calculated:time to maximum plasma concentration (T_(max)), maximum plasmaconcentration (C_(max)), maximum plasma concentration extrapoloated totime 0 (C₀), area under the plasma concentration-time curve to the lasttime point (AUC_(last)) and to infinity (AUC_(int)).

Abbreviations Used: ATCC, American Type Culture Collection: BTO,1,2,4-benzotriazine di-N-oxides; CC₅₀, cytotoxicity concentration;CDCl₃, deuterated chloroform; CD₃OD, deuterated methanol; CFU, colonyforming unit; CLSI, Clinical and Laboratory Standards Institute; CSI.Colorado State University; DOTS, direct observed therapy short-course;E_(1/2), one electron reduction potential; ES-MS, electrospray massspectra; FBS, fetal bovine serum; LC-MS-MS, liquid chromatograph tandemmass spectrometer; LCQ, liquid chromatography quadrupole; LORA,low-oxygen recovery assay; MDR-TB, multidrug-resistant TB; MEM, minimalessential medium; MLM, mouse liver microsome; MOLY, mouse lymphoma cellmutation assay; Mtb, Mycobacterium tuberculosis; NRP, nonreplicatingpersistence; RLU, relative light unit; SD, standard deviation; SI,selectivity index; TB, tuberculosis; TFAA, trifluoroacetic anhydride;TI, therapeutic index; TPZ, tirapazamine; XDR, extensivelydrug-resistant.

Supporting Information Table 1. Spectrum of antimicrobial activity forBTOs. 8a 20q Bacterial strains Source (μg/mL) (μg/mL) Mtb H37Rv CSU 1.20.31 Mycobacterium smegmatis ATCC 10143 5.0 2.5 Mycobacterium abscessusATCC 19977 >32 >32 Klebsiella pneumoniae ATCC 700721 >32 >32 Klebsiellapneumoniae ATCC 43816 >32 >32 Enterobacter aerogenes ATCC 29751 >32 >32Enterobacter aerogenes SRI Collection >32 >32 Escherichia coli BAA 20116 >32 Escherichia coli ATCC 25922 16 >32 Acinetobacter baumannii BAA1605 >32 >32 Acinetobacter baumannii ATCC 17978 32 >32 Staphylococcusaureus ATCC 29213 >32 >32 Staphylococcus aureus HA-MRSA NRS 38216-32 >32 Enterococcus fecalis ATCC 29212 16 >32 Enterococcus fecalisATCC 51575 32 >32 Escherichia coli pKM101 SRI Collection 32 >32Salmonella typhimurium-Hisg46 SRI Collection >32 >32 Salmonellacholeraesuis ATCC 13311 32 >32 Pseudomonas aeruginosa ATCC 27853 >32 >32Enterococcus faecium ATCC 19434 >32 >32 Moraxella catarrhalis ATCC 2523832 10

Experimental Method for the Salmonella/Microsome Plate IncorporationAssay (Ames Screen).

Experimental compounds were screened for microbial mutagenicity activityusing the plate incorporation method with Salmonella typhimurium testerstrains TA98 and TA100, in the presence and absence of an Aroclor1254-induced rat-liver metabolic activation system containing 10% S9(MA). The presence of the appropriate genetic characteristics wasverified for the strains used in this study. Stock solutions were madeby dissolving each test sample in dimethyl sulfoxide (DMSO) at 5 mg/ml.These initial stock concentrations were then serially diluted to achievedose formulations of 100, 50, 10, 5, 1, 0.5, and 0.1 μg/plate (dosingvolume 100 μl). Test plates were compared with the control plates fortheir revertant count and for the condition of the background bacteriallawn. A test article was considered a mutagen when the mean number ofrevertant colonies on the test plates exceeds the mean solvent controlcounts by at least a two-fold margin. Dose relatedness was also takeninto account when evaluating a mutagenic response. Cytotoxicity wasestimated by several parameters: a substantial decrease in the number ofrevertant colonies on the test plates, clearing or absence of thebackground bacterial lawn growth, formation of pinpoint nonrevertantcolonies, or complete absence of bacterial growth.

Supporting Information Table 2. Evaluation of 15fa and 20q in theSalmonella/Microsome Plate Incorporation Assay (Ames Screen).

Data for Positive Controls:

Mean Ratio Dose level revertants Standard treated/ Individual revertantStrain Compound per plate per plate Deviation solvent colony countsWithout metabolic activation TA98 2NF 5 μg 1230.7 94.6 48.3 1247, 1129,1316 TA100 SA 5 μg 1620.0 32.6 10.0 1589, 1617, 1654 TA98 DMSO — 20.33.8 0.8 16, 22, 23 TA100 DMSO — 157.3 10.2 1.0 169, 153, 150 Withmetabolic activation (10% S9) TA98 2AN (10% S9) 2μg 543.3 20.0 15.3 528,536, 566 TA100 2AN (10% S9) 2μg 490.0 37.7 2.9 532, 459, 479 TA98 DMSO(+S9) — 30.3 10.4 0.9 27, 22, 42 TA100 DMSO (+S9) — 174.7 21.5 1.0 158,167, 199 Key to Positive Controls Key to Plate Postfix Codes 2NF2-Nitrofluorene SA Sodium Azide 2AN (10% S9) 2-Aminoanthracene (10% S9)DMSO (+S9) Dimethyl Sulfoxide +S9

Data for 15fa:

Data for 15fa: Mean Ratio Dose level revertants Standard treated/Individual revertant Strain Compound per plate per plate Deviationsolvent colony counts Without metabolic activation TA98 15fa 0.1 μg 21.01.4 0.8 22, 20 15fa 0.5 μg 19.0 4.2 0.7 22, 16 15fa 1 μg 26.0 5.7 1.030, 22 15fa 5 μg 47.0 2.8 1.8 45, 49 15fa 10 μg 45.0 5.7 1.8 41, 49 15fa50 μg 141.0 45.3 5.5 109, 173 N 15fa 100 μg 215.5 9.2 8.5 222 N, 209 NDMSO 25.5 0.7 26, 25 Untreated Control 22.2 5.5 26, 27, 14, 25, 19 TA10015fa 0.1 μg 127.0 5.7 0.8 131, 123 15fa 0.5 μg 137.0 1.4 0.8 138, 13615fa 1 μg 150.5 2.1 0.9 152, 149 15fa 5 μg 259.0 8.5 1.6 253, 265 15fa10 μg 299.5 33.2 1.8 323, 276 15fa 50 μg 913.5 99.7 5.6 984, 843 N 15fa100 μg 1268.0 271.5 7.8 1076 N, 1460 N DMSO 162.5 44.5 194, 131Untreated Control 165.2 12.0 149, 172, 177, 172, 156 With metabolicactivation (10% S9) TA98 15fa 0.1 μg 35.5 6.4 1.0 31, 40 15fa 0.5 μg36.0 1.4 1.0 37, 35 15fa 1 μg 30.5 0.7 0.9 31, 30 15fa 5 μg 37.0 0.0 1.037, 37 15fa 10 μg 52.0 0.0 1.5 52, 52 15fa 50 μg 142.5 6.4 4.0 138, 147N 15fa 100 μg 345.0 39.6 9.7 317 N, 373 N DMSO 35.5 2.1 37, 34 TA10015fa 0.1 μg 145.5 0.7 0.9 146, 145 15fa 0.5 μg 148.5 4.9 0.9 152, 14515fa 1 μg 185.0 8.5 1.1 179, 191 15fa 5 μg 207.0 12.7 1.2 198, 216 15fa10 μg 298.5 37.5 1.8 272, 325 15fa 50 μg 854.5 30.4 5.1 833, 876 N 15fa100 μg 1450.0 12.7 8.6 1441 N, 1459 N DMSO 168.5 12.0 160, 177 Key toPositive Controls Key to Plate Postfix Codes N Normal background lawn

Data for 20q:

Mean Ratio Dose level revertants Standard treated/ Individual revertantStrain Compound per plate per plate Deviation solvent colony countsWithout metabolic activation TA98 20q 0.1 μg 33.0 2.8 1.0 31 N, 35 N 20q0.5 μg 50.5 2.1 1.6 52 N, 49 N 20q 1 μg 85.0 5.7 2.7 81 N, 89 N 20q 5 μg241.0 18.4 7.5 228 N, 254 N 20q 10 μg 378.5 89.8 11.8 315 N, 442 N 20q50 μg 797.0 4.2 24.9 800 N, 794 N 20q 100 μg 1133.0 32.5 35.4 1110 N,1156 N DMSO 32.0 5.2 38, 29, 29 Untreated Control 33.6 8.0 42, 37, 38,29, 22 TA100 20q 0.1 μg 173.0 8.5 1.2 179 N, 167 N 20q 0.5 μg 155.0 14.11.0 165 N, 145 N 20q 1 μg 206.0 4.2 1.4 203 N, 209 N 20q 5 μg 303.0 25.52.0 321 N, 285 N 20q 10 μg 392.0 36.8 2.6 366 N, 418 N 20q 50 μg 1033.550.2 7.0 998 N, 1069 N 20q 100 μg 1468.0 29.7 9.9 1489 N, 1447 N DMSO148.3 8.1 154, 139, 152 Untreated Control 143.4 12.6 153, 146, 123, 154,141 With metabolic activation (10% S-9) TA98 20q 0.1 μg 39.5 3.5 1.4 42N, 37 N 20q 0.5 μg 30.0 0.0 1.1 30 N, 30 N 20q 1 μg 47.5 2.1 1.7 46 N,49 N 20q 5 μg 124.0 9.9 4.5 117 N, 131 N 20q 10 μg 266.5 10.6 9.8 259 N,274 N 20q 50 μg 988.0 4.2 36.1 991 N, 985 N 20q 100 μg 1566.0 82.0 57.31508 N, 1624 N DMSO 27.3 6.1 34, 22, 26 TA100 20q 0.1 μg 171.5 31.8 1.1149 N, 194 N 20q 0.5 μg 204.0 1.4 1.3 205 N, 203 N 20q 1 μg 180.5 4.91.1 177 N, 184 N 20q 5 μg 267.0 8.5 1.7 273 N, 261 N 20q 10 μg 291.514.8 1.8 302 N, 281 N 20q 50 μg 700.5 23.3 4.4 684 N, 717 N 20q 100 μg916.0 21.2 5.8 901 N, 931 N DMSO 157.7 4.5 162, 158, 153 Key to PositiveControls Key to Plate Postfix Codes DMSO Dimethyl Sulfoxide N Normalbackground lawn

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What is claimed is:
 1. A compound having the structure of formula I:

wherein: each X is independently H, halogen, alkyl, OR, SR, NR′R, orBR′R, wherein each R and each R′ is independently H, halogen, or alkyl;W is N; each A and B is optionally substituted alkyl, or A and B arejoined in an optionally substituted heterocycloalkyl; and Z is anoptionally present, optionally substituted 5 or 6-membered ring,saturated or unsaturated, fused to the bezotriazine ring at the 6,7position, or a pharmaceutically acceptable salt or stereoisomer thereof.2. The compound of claim 1 wherein: each X is H; W is N; A and B arejoined in an optionally substituted piperidinyl or pyrrolidinyl; and Zis present as a 5-membered ring, fused to the bezotriazine ring at the6,7 position, or a pharmaceutically acceptable salt or stereoisomerthereof.
 3. The compound of claim 1 selected from:3-(diethylamino)benzo[e][1,2,4]triazine 1,4-dioxide (17o);3-morpholinobenzo[e][1,2,4]triazine 1,4-dioxide (17p);3-(pyrrolidin-1-yl)benzo[e][1,2,4]triazine 1,4-dioxide (17q);3-(pyrrolidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20q);3-(piperidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20r);3-(dimethylamino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20s);3-(benzyl(methyl)amino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20t);3-(methyl(phenethyl)amino)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20u);3-(thiomorpholinosulfone-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20v);3-(2-methylpiperidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20w); and3-(4-(methylsulfonyl)piperazin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20x), or a pharmaceutically acceptable salt or stereoisomerthereof.
 4. The compound of claim 1 that is:3-(pyrrolidin-1-yl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine1,4-dioxide (20q), or a pharmaceutically acceptable salt or stereoisomerthereof.
 5. A pharmaceutical composition or kit comprising a compound ofclaim 1 and a second, different anti-mycobacterium tuberculosis (Mtb)drug.
 6. A pharmaceutical composition or kit comprising a compound ofclaim 2 and a second, different anti-mycobacterium tuberculosis (Mtb)drug.
 7. A method of making a compound of claim 1 according to thefollowing scheme:

(a) HOF.ACN, DCM; b) HNR₂R₃, Et₃N, DCM; wherein X is R₁, W is N, A is R₃and B is R₂.
 8. A method of making a compound of claim 2 according tothe following scheme:

(a) HOF.ACN, DCM; b) HNR₂R₃, Et₃N, DCM; wherein X is R₁, W is N, A is R₃and B is R₂.
 9. A method of treating a mycobacterium tuberculosis (Mtb)infection, comprising: contacting a person in need thereof with aneffective amount of compound of claim
 1. 10. A method of treating amycobacterium tuberculosis (Mtb) infection, comprising: contacting aperson in need thereof with an effective amount of compound of claim 2.